MAIMOTOMONTANT US009944986B2 DIU TO DI UN ANONLINE (12 ) United States Patent (10 ) Patent No. : US 9 , 944 , 986 B2 Nagy (45 ) Date of Patent: Apr . 17 , 2018
( 54 ) THERAPEUTIC TARGETS FOR Hirschhorn et al. (Genetics in Medicine. vol. 4 , No. 2 , pp . 45- 61 , ALZHEIMER ' S DISEASE Mar. 2002 ) . * Lucentini et al ( The Scientist ( 2004 ) vol. 18 , p . 20 ) . * ( 71 ) Applicant: The University of Birmingham , Langdahl ( Journal of Bone and Mineral Research 2000 vol . 15 , No. 3 , pp . 402 -414 ). * Birmingham (GB ) Wall (Nature Reviews Genetics ( 2003) vol. 4 , pp . 587 -597 ). * (72 ) Inventor : Zsuzsanna Nagy , Birmingham (GB ) Abramsson et al. , “ No Association of LOXLI Gene ( 73 ) Assignee : The University of Birmingham , Polymprophisms with Alzheimer ' s Disease ” , Neuromol. Med . , 13 : 160 - 166 ( 2011 ) . Birmingham , West Midlands (GB ) Bonda et al. , “ Novel therapeutics for Alzheimer' s disease : An ( * ) Notice : Subject to any disclaimer , the term of this update ” , Curr. Opin . Drug Discov . Devel. , 13 ( 2 ) : 235 - 246 ( 2010 ) . patent is extended or adjusted under 35 Bove et al. , “ Fighting neurodegeneration with rapamycin : mecha nistic insights ” , Nature Reviews, Neuroscience , 12 : 437 -452 (2011 ) . U . S . C . 154 (b ) by 101 days . Caccamo et al. , “ Molecular Interplay between Mammalian Target of ( 21 ) Appl . No. : 14 /413 , 659 Rapamycin (mTOR ) , Amyloid - B , and Tau ” , J . Biol. Chem . , 285 ( 17 ) : 13107 - 13120 ( 2010 ) . (22 ) PCT Filed : Jul. 11 , 2013 Cai et al. , “ Mammalian Target of Rapamycin : A Valid Therapeutic Target Through the Autophagy Pathway for Alzheimer ' s Disease ? ” , ( 86 ) PCT No .: PCT/ GB2013 / 051843 Journal of Neuroscience Research , 90 : 1105 - 1118 (2012 ). Cascalheira et al . , " Serum homocysteine : Interplay with other $ 371 ( c )( 1 ) , circulating and genetic factors in association to Alzheimer ' s type ( 2 ) Date : Jan . 8 , 2015 dementia” , Clinical Biochemistry , 42 : 783 - 790 (2009 ). Clifford et al ., “ Large - scale analysis of non - synonymous coding ( 87 ) PCT Pub . No. : WO2014 /009733 region single nucleotide polymorphisms” , Bioinformatics, 20 ( 7 ) : 1006 - 1014 ( 2004 ) . PCT Pub . Date : Jan . 16 , 2014 Erikkson et al. , “ Associations of gene sequence variation and serum levels of C -reactive protein and Interleukin - 6 with Alzheimer ' s (65 ) Prior Publication Data disease and dementia ” , J . Alzheimers Dis . , 23 ( 2 ) : 361 - 369 ( 2011 ) . Finckh , “ The Future of genetic association studies in Alzheimer US 2015/ 0141491 A1 May 21 , 2015 disease ” , J . Neural. Transm , 110 : 253 - 266 ( 2003 ) . Forlenza et al. , “ Diagnosis and biomarkers of predementia in Foreign Application Priority Data Alzheimer ' s disease ” , BMC Medicine , 8 : 89 (2010 ) . ( 30 ) Gustaw - Rothenberg et al ., “ Biomarkers in Alzheimer 's disease : 1212334 . 5 past , present and future ”, Biomark . Med ., 4 ( 1) : 15 - 26 (2010 ). Jul . 11 , 2012 (GB ) .. . Heinonen et al. , “ Deciphering downstream gene targets of P13K / mTOR /p70S6K pathway in breast cancer” , BMC Genomics, 9 : 348 (51 ) Int. Ci. ( 2008 ) . C12Q 1 /68 ( 2006 . 01 ) Hong et al. , “ Genome- Wide and Gene- Based Association Impli C12N 15 / 113 ( 2010 . 01) cates FRMD6 in Alzheimer Disease” , Human Mutation , 33 ( 3 ) : ( 2006 .01 ) 521 - 529 (2012 ) . A61K 31/ 436 Flex et al ., “ Polymorphisms of the Macrophage Inhibitory Factor GOIN 33 / 50 ( 2006 .01 ) and C -Reactive Protein Genes in Subjects with Alzheimers Demen (52 ) U . S . CI. tia ” , Dement . Geriatr. Cogn . Disord ., 18 : 261 -264 ( 2004 . CPC ...... C12Q 1/ 6883 (2013 . 01 ) ; A61K 31/ 436 Kok et al. , “ CRP gene variation affects early development of ( 2013 .01 ) ; C12N 15 / 113 (2013 .01 ) ; GOIN Alzheimer ' s disease -related plaques” , Journal of Neuroinflamma tion , 8 : 96 (2011 ) . 33 /5023 (2013 .01 ) ; C12N 2310 / 14 (2013 .01 ) ; Kramer et al. , “ Alzheimer disease pathology in cognitively healthy C120 2600 / 106 ( 2013 .01 ) ; C12Q 2600 / 118 elderly : A genome- wide study” , Neurobiology of Aging , 32 : 2113 (2013 . 01 ) ; C12Q 2600/ 136 (2013 . 01 ) ; C12Q 2122 ( 2011 ) . 2600 / 156 (2013 .01 ) ; C12Q 2600 / 158 Lesseux et al. , “ Syk -dependent mTOR activation in follicular (2013 .01 ) lymphoma cells ” , Blood , 108 ( 13 ) : 4156 -4162 ( 2006 ) . Li et al. , “ Levels of mTOR and its downstream targets 4E -BP1 , (58 ) Field of Classification Search eEF2 , and eEF2 kinase in relationships with tau in Alzheimer' s None disease brain ” , FEBS Journal , 272 : 4211 -4220 (2005 ) . See application file for complete search history. Liu et al. , “ A Genomewide Screen for Late -Onset Alzheimer Disease in a Genetically Isolated Dutch Population ” , The American ( 56 ) References Cited Journal of Human Genetics, 81: 17 -31 ( 2007) . U . S . PATENT DOCUMENTS (Continued ) Primary Examiner — Katherine D Salmon 2002/ 0015941 Al * 2 / 2002 Kim ...... GOIN 33 /5008 (74 ) Attorney , Agent, or Firm — Kathleen D . Rigaut; 435 / 4 2003 /0092019 A1 * 5 / 2003 Meyer ...... CO7K 14 / 47 Dann , Dorfman , Herrell and Skillman , P . C . 435 / 6 . 14 (57 ) ABSTRACT The present invention relates to novel methods for the FOREIGN PATENT DOCUMENTS prevention , treatment and diagnosis of Alzheimer ' s disease . In addition , the invention relates to methods for assessing an WO 2011/ 012672 A1 2 /2011 individual' s susceptibility or pre - disposition to Alzheimer ' s disease . The methods of the present invention involve the use of therapeutic targets and diagnostic and /or predictive OTHER PUBLICATIONS markers within the mTOR signalling pathway . The methods Morita ( AJH 2006 vol. 19 pp . 593 -600 ). * also involve screening subjects for genetic polymorphisms Bertram ( The American Journal of Human Genetics Nov . 7 , 2008 associated with rapamycin - sensitive genes . vol. 83 pp . 623 -632 ). * 4 Claims, 34 Drawing Sheets US 9 ,944 , 986 B2 Page 2
References Cited Ueberham et al. , “ Altered subcellular location of phosphorylated ( 56 ) SMads in Alzheimer' s disease” , European Journal of Neuroscience , 24 : 2327 - 2334 (2006 ) . OTHER PUBLICATIONS Oijen et al ., “ Polymorphisms and haplotypes in the C -reactive McKhann et al. , “ Clinical diagnosis of Alzheimer' s disease: Report protein gene and risk of dementia ” , Neurobiology of Aging , 28 : 1361- 1366 ( 2007 ) . of the NINCDS- ADRDA Work Group * under the auspices of Xiao et al. , “ Lysyl Oxidase, Extracellular Matrix Remodeling and Department ofHealth and Human Services Task Force on Alzheim Cancer Metastasis” , Cancer Microenvironment, 5 : 261- 273 (2012 ) . er' s Disease ” , Neurology , 34 : 939 - 944 ( 1984 ). Yates et al. , “ Dysfunction of the mTOR pathway is a risk factor for Mendelsohn et al. , “ Rapamycin As an Antiaging Therapeutic ? : Alzheimer ' s disease ” , Acta Neuropathologica Communications, 1 : Targeting Mammalian Target of Rapamycin to Treat Hutchinson 3 ( 2013 ) . Gilford Progereia and Neurodegenerative Diseases” , Rejuvenation Yates et al. , “ Role of the Proliferation -Related Molecules in the Research , 14 ( 4 ): 437 -441 (2011 ). Pathogenesis of Alzheimer ' s Disease ” , A Thesis submitted to the Morita et al . , “ Association Study Between C - Reactive Protein University of Birmingham for the degree of Master of Philosophy , Genes and Ischemic Stroke in Japanese Subjects ” , AJH , 19 : 593 pp . 1 -332 , Apr. 2012 . 600 (2006 ) . Zemke et al. , “ The mTOR Pathway as a Potential Target for the Morita et al. , “ Polymorphism of the C -Reactive Protein (CRP ) Gene Development of Therapies Against Neurological Disease ” , Drug is Related to Serum CRP Level and Arterial Pulse Wave Velocity in News Perspect , 20 ( 8 ) : 495 -499 (2007 ) . Healthy Elderly Japanese ” , Hypertens. Res. , 29 : 323 -331 (2006 ) . Genolet et al ., “ An approach to analyse the specific impact of Nagy, “ The dysregulation of the cell cycle and the diagnosis of rapamycin on mRNA -ribosome association ” , BMC Medical Alzheimer ' s disease” , Biochimica et Biophysica Acta , 1772 : 402 Genomics, 1 : 33 ( 2008 ) . 408 ( 2007 ) . Search Report, dated Aug. 1 , 2016 , issued in corresponding Euro Oddo , “ The role of mTOR signaling in Alzheimer ' s disease” , pean Patent Application No. 13 740 046 . 1 . Frontiers in Bioscience, S4 : 941 - 952 (2012 ) . International Search Report /Written Opinion , dated Jan . 15 , 2014 , Perry et al. , " A New Alzheimer ' s Disease Interventive Strategy : issued in corresponding PCT/GB2013 / 051843 . GLP- 1 ” , Current Drug Targets, 5 : 565 -571 (2004 ) . Biesecker , Leslie G . et al ., “ The ClinSeq Project : Piloting large Perry et al. , “ Enhancing Central Nervous System Endogenous scale genome sequencing for research in genomic medicine ” , GLP - 1 Receptor Pathways for Intervention in Alzheimer' s Dis Genome Research , 19 ( 9 ) : 1665 - 1674 (2009 ) . ease ” , Current Alzheimer Research , 2 : 377 - 385 (2005 ) . Connelly, Jessica J . et al. , “ Genetic and functional association of Santos et al. , “ Effects of rapamycin and TOR on aging and memory : FAM5C with myocardial infarction ” , BMC Medical Genetics, 9 : 33 implications for Alzheimer 's disease” , Journal of Neurochemistry , ( 2008 ) . 117 : 927 -936 ( 2011) . European Examination Report , dated May 3 , 2017 , issued in cor Sun et al ., " Protective Effects of Bone Morphogenetic Protein 7 responding European Patent Application No . 13740046 . 1 . Against Amyloid - Beta Induced Neurotoxicity in PC12 Cells ” , Neu roscience , 184 : 151 - 163 (2011 ). * cited by examiner U. S . Patent atent Apr . 17 , 2018 Sheet 1 of 34 US 9, 944 , 986 B2
Fig 1
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Fig 2
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Fig 3 continued D - tau content in whole population
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Fig 4
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Fig 8 G1 cells ( % )
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Fig 11 Prpt pg? } whole population
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Fig 13 continued 1 . , 1 . , 1 , 2 , . , . , . , . , . , . , Protein per cell whole population
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Fig 13 continued
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Fig 15 continued p - tau per cell in whole population 150
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Fig 15 continued
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Fig 16A
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Fig 16B U . S . Patent Apr . 17 , 2018 Sheet 32 of 34 US 9 ,944 , 986 B2
Fig 16C E 0 atent Apr . 17 , 2018 Sheet 33 of 34 US 9 ,944 , 986 B2
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W US 9 , 944 , 986 B2 THERAPEUTIC TARGETS FOR In addition to difficulties associated with diagnosing ALZHEIMER ' S DISEASE Alzheimer ' s disease , there are also problems associated with identifying individuals in the population who are at This application is a $ 371 national phase entry of Inter increased and / or decreased risk of developing Alzheimer ' s national Patent Application PCT GB2013/ /051843 , filed 11 5 disease during their lifetime, as compared with the average Jul. 2013 , which claims priority to GB Patent Application level of risk associated with the general population . The only No . 1212334 . 5 filed Jul. 11 , 2012 , the entire contents of each known genetic risk factor for late onset sporadic form of being incorporated by reference herein as though set forth in Alzheimer ' s disease is the polymorphism on the ApoE gene . Other discovered polymorphisms appear to be restricted to full . 10 relatively small patient subgroups . Thus risk prediction or FIELD OF THE INVENTION assessment of susceptibility , before the development of clinical Alzheimer ' s disease , is difficult as well . There is now good evidence to suggest that the neuropa The present invention relates to novel strategies for the thology underlying Alzheimer' s disease begins years , maybe prevention , treatment and diagnosis of Alzheimer ' s disease . 15 even a decade , prior to the diagnosis of clinical dementia In addition , the invention relates to strategies for assessing ( Forlenza et al. , ( 2010 ) BMC Medicine 8 : 89 ) . Based on an individual' s susceptibility or pre - disposition to Alzheim these observations , the continuum of Alzheimer ' s disease er 's disease . In particular , the present invention relates to progression has been classified into three phases : methods involving the use of therapeutic targets and diag ( i ) asymptomatic Alzheimer ' s disease (preclinical stage ) ; nostic and / or predictive markers within the mTOROR signalSignal- 2020 (G ii ) mild cognitive impairment (MCI ) due to Alzheimer 's ling pathway . disease ( pre - dementia stage ) ; and ( iii ) clinically - defined Alzheimer ' s disease (dementia ) . BACKGROUND TO THE INVENTION In light of the above , there now exist several opportunities for improved management of Alzheimer ' s disease . In par Alzheimer ' s disease is the most common form of demen - 25 ticular, itmay be possible to identify individuals at increased tia in older people . As a result of population aging world - risk of developing Alzheimer 's disease , and /or diagnose wide , the prevalence of this disease is set to increase individuals at a much earlier stage of disease , for example , significantly in coming years . As such , there is an urgent individuals with asymptomatic disease or those patients with need to develop better prognostic and diagnostic tools and MCI that will go on to develop clinically - defined Alzheim new treatments for people identified as having this disease . 30 er ' s disease . If susceptible individuals can be identified Alzheimer ' s disease is a chronic neurodegenerative dis - and / or diagnosed at an earlier stage of disease , it will be order characterised by selective loss of cortical neurons possible to develop , test and use new preventative and / or within the hippocampus and the temporal and frontal lobes curative treatments intended to stabilize and / or reverse the of the brain . The neurodegenerative process occurring in neurodegenerative process and thereby prevent cognitive Alzheimer ' s disease is accompanied by progressive cogni- 35 decline . tive impairment leading ultimately to dementia in affected Researchers are already using the improved knowledge of individuals . Alzheimer' s disease pathogenesis to develop more effective There is currently no accepted “ gold standard ” diagnostic methods of diagnosis and treatment. In this regard , diagnos test for Alzheimer 's disease in the live patient. This reflects tic biomarkers have been identified that can be measured in the difficulties associated with identifying patients who 40 humoral fluids, mainly cerebrospinal fluids, and biomarkers would go on to be classified as having this disease at post that may be detected using advanced neuroimaging methods mortem examination . Clinical diagnosis of Alzheimer ' s dis (Gustaw -Rothenberg et al. , (2010 ) Biomark . Med . 4 ( 1 ): 15 ease is typically based on evaluation of clinical criteria , such 26 ). as the NINCDS /ADRDA criteria (McKhann , G . et al. , Furthermore , new “ disease -modifying ” treatments are ( 1984 ) Neurology 34 : 939 - 944 ). 45 being developed that tackle the deposition of ß - amyloid The problem with the diagnostic methods used to date lies plaques and NFTs ( Bonda et al ., (2010 ) Curr . Opin . Drug in the fact that patients are typically diagnosed once clinical Discov Devel. 13 ( 2 ) : 235 - 246 ) dementia has started to develop . It follows therefore , that There remains however, an ongoing need to improve existing treatment strategies are limited to agents used methods for the diagnosis and treatment of Alzheimer' s primarily to manage the symptoms of disease . For example , 50 disease , particularly early - stages of disease . The present cholinesterase inhibitors are administered to patients so as to invention seeks to address these issues . block the degradation of the neurotransmitter acetylcholine and thereby enhance neurotransmission in the brain . Use of SUMMARY OF INVENTION such agents can help to preserve cognitive function , but does not improve the underlying pathology and is therefore not a 55 The present invention is directed towards new methods of curative approach . preventing, treating and diagnosing Alzheimer' s disease Although the etiology of Alzheimer ' s disease is poorly based on the use of novel gene targets linked to this disease . understood , the neuropathology associated with the devel The invention also relates to screening methods for identi opment ofthis disease has been relatively well characterised . fying individuals that are pre - disposed to Alzheimer ' s dis The classical hallmarks of this disease consist of amyloid - ß 60 ease , based on the use of novel gene targets . plaques, which accumulate in the brain , and neurofibrillary The gene targets presented herein are classified as tangles (NFT ) consisting ofhyperphosphorylated tau protein " rapamycin -sensitive ” genes for the reason that their cellular present in affected neurons. Additional changes occurring at expression is affected by the compound rapamycin . The the cellular level, which are now thought to precede the group of rapamycin - sensitive genes to which the present deposition of plaques and NFTs, include damage to cells 65 invention relates were found to be deregulated in the brains caused by oxidative stress , mitochondrial malfunction and of patients diagnosed with Alzheimer ' s disease , as compared aberrant re - entry of neurons into the cell division cycle . with control samples, using microarray expression analysis . US 9 , 944 ,986 B2 Rapamycin is known to inhibit the serine/ threonine kinase and 5 , and /or the activity of one ormore of the proteins mTOR and thereby reduce signalling downstream of this encoded by the rapamycin -sensitive genes shown in protein therefore the present invention is directed in par Tables 2 , 3 , 4 and 5 ; ticular , to the use of novel therapeutic and diagnostic targets ( ii ) comparing the expression level and / or activity mea within the mTOR signalling pathway in the context of 5 sured in ( i ) with reference / control values , methods for the prevention , treatment and diagnosis of wherein a difference in expression level and / or activity of Alzheimer ' s disease . the one or more rapamycin sensitive genes shown in Tables In a first aspect , the present invention provides a method 2 , 3 , 4 and 5 is indicative of Alzheimer ' s disease progres sion . for the prevention and /or treatment of Alzheimer 's disease in 10 In a fifth aspect , the invention provides a method for a subject , comprising administering to the subject a phar screening a human subject for pre - disposition to Alzheim macological agent which modulates one or more targets er ' s disease , which method comprises the steps of: within the mTOR signalling pathway of a cell, wherein the ( i ) measuring either the expression level of one or more of target is selected from : the rapamycin -sensitive genes shown in Tables 2 , 3 , 4 ( i) the rapamycin - sensitive genes shown in Tables 2 , 3 , 4 15 . and 5 , and / or the activity of one or more of the proteins and 5 ; encoded by the rapamycin - sensitive genes shown in ( ii ) the transcriptional products of the rapamycin -sensitive Tables 2 , 3 , 4 and 5 ; genes shown in Tables 2 , 3 , 4 and 5 , or fragments ( ii ) comparing the expression level and / or activity mea thereof; and sured in ( i ) with reference /control values, (iii ) the proteins encoded by the rapamycin -sensitive 20 wherein a difference in expression level and / or activity of genes shown in Tables 2 , 3 , 4 and 5 , or fragments the one or more rapamycin sensitive genes shown in Tables thereof. 2 , 3 , 4 and 5 is taken to mean the subject is pre - disposed to In a second aspect, the invention provides a method of Alzheimer ' s disease. screening for pharmacological agents useful in the preven In all aspects of the invention described above , in pre tion and / or treatment of Alzheimer ' s disease in a subject, 25 ferred embodiments , the one or more targets within the wherein said method comprises: mTOR signalling pathway is / are selected from the group of ( i) contacting a cell with a test pharmacological agent; ranamrapamycin - sensitive genes consisting of calcium channel , ( ii ) measuring either the expression level of one or more voltage -dependent , L type, alpha 1D subunit (CACNA1 D ) , of the rapamycin -sensitive genes shown in Tables 2 , 3 , gamma - aminobutyric acid B receptor, 2 (GABBR2 ) , 4 and 5 , or the level or activity of one or more of the 30 homeobox D10 (HOXD10 ) , Kruppel- like factor 2 (KLF2 ), proteins encoded by the rapamycin - sensitive genes rhodopsin (RHO ) and GLI zinc finger family 2 (GLI2 ) . shown in Tables 2 , 3 , 4 and 5 ; The present invention is also directed to methods for ( iii ) measuring either the expression level of one or more identifying human subjects that are pre - disposed to of the rapamycin - sensitive genes shown in Tables 2 , 3 , Alzheimer ' s disease , and methods to assist with diagnosis of 4 and 5 , or the level or activity of one or more of the 35 Alzheimer ' s disease in live human subjects based on the use proteins encoded by the rapamycin - sensitive genes of polymorphisms, particularly single nucleotide polymor shown in Tables 2 , 3 , 4 and 5 in a control cell not phisms ( SNPs ), in the rapamycin -sensitive genes described exposed to the test pharmacological agent; herein . Methods are also described based on the use of ( iv ) comparing the results determined in steps ( ii ) and ( iii ) polymorphisms within genes which affect the expression of wherein a difference in the expression level of one or 40 rapamycin - sensitive genes . more of the rapamycin -sensitive genes shown in Tables Therefore , in a sixth aspect, the invention provides a 2 , 3 , 4 and 5 , or the level or activity of one or more of method of screening a human subject for pre -disposition to the proteins encoded by the rapamycin -sensitive genes Alzheimer ' s disease, which method comprises genotyping shown in Tables 2 , 3 , 4 and 5 , indicates that the test the subject for one or more polymorphisms in one or more pharmacological agent is suitable for use in the pre - 45 of the rapamycin - sensitive genes shown in Table 1 , wherein vention and /or treatment of Alzheimer 's disease. the presence of at least one variant allele associated with In a third aspect , the invention provides a method to assist Alzheimer ' s disease is an indication that the subject is with diagnosis of Alzheimer ' s disease in a live human pre - disposed to Alzheimer ' s disease . subject, which method comprises the steps of: - . In a seventh aspect, the invention provides a method of ( i ) measuring either the expression level of one or more of 50 screening a human subject for pre - disposition to Alzheim the rapamycin - sensitive genes shown in Tables 2 , 3 , 4 er ' s disease , which method comprises genotyping the sub and 5 , and /or the activity of one ormore of the proteins ject for one or more polymorphisms in one or more genes encoded by the rapamycin - sensitive genes shown in which affect the expression of one or more of the rapamycin Tables 2 , 3 , 4 and 5 ; sensitive genes shown in Table 1 , wherein the presence of at ( ii ) comparing the expression level and / or activity mea - 55 least one variant allele associated with Alzheimer 's disease sured in ( i ) with reference / control values, is an indication that the subject is pre - disposed to Alzheim wherein a difference in expression level of one or more of e r' s disease . the rapamycin - sensitive genes shown in Tables 2 , 3 , 4 In an eighth aspect , the invention provides a method to and 5 , and/ or the level or activity of one or more of the assist with diagnosis of Alzheimer ' s disease in a live human proteins encoded by the rapamycin - sensitive genes 60 subject, which method comprises genotyping the subject for shown in Tables 2 , 3 , 4 and 5 is indicative of Alzheim - one or more polymorphisms in one or more of the rapamy er 's disease . cin - sensitive genes shown in Table 1 , or a polymorphism in In a fourth aspect , the invention provides a method of one or more genes which affect the expression of one or assessing the risk of Alzheimer ' s disease progression in a more of the rapamycin -sensitive genes shown in Table 1 , human subject, which method comprises the steps of: - 65 wherein the presence of at least one variant allele associated ( i ) measuring either the expression level of one or more of with Alzheimer' s disease is indicative of Alzheimer ' s dis the rapamycin -sensitive genes shown in Tables 2 , 3 , 4 ease. US 9 , 944 , 986 B2 un In a ninth aspect, the invention provides an array or kit for treated with Culture medium , siRNA Control and CACNA1 detecting genetic polymorphisms in a sample taken from a D siRNA alone ; Darker shading = cells treated with addi subject wherein the array or kit comprises reagents for the tional 100 ng /ml rapamycin . detection of one or more polymorphisms in one or more of FIG . 5 Effect of siRNA -mediated down -regulation of the rapamycin - sensitive genes shown in Table 1 , or one or 5 CACNA1 D on p - tau expression in SH -SY5Y neuroblas more polymorphisms in one or more genes which affect the toma cells. Grey bars = all single cells ; Vertically - shaded expression of one or more of the rapamycin - sensitive genes bars = G1 population ; Horizontally - shaded bars = G2 popula shown in Table 1 . tion ; Lighter shading = cells treated with Culture medium , The present invention is also concerned with polymor siRNA Control and CACNA1 D siRNA alone ; Darker phisms that may be used to monitor the mTOR signalling 10 shades represent cells treated with additional 100 ng /ml pathway in a cell . These polymorphisms are associated with rapamycin . All data are normalised to Control ( 100 % ) . the differential sensitivity of cells to the G1/ S inhibitor FIG . 6 Effect of siRNA -mediated down -regulation of rapamycin . GABBR2 on cell cycle kinetics in SH - SY5Y neuroblastoma Therefore , in a further aspect, the invention provides a cells . Vertically - shaded bars = G1 population ; Horizontally method by which to monitor mTOR signalling in a human 15 shaded bars = G2 population ; Lighter shading = cells treated cell, which method comprises detecting one or more poly with Culture medium , siRNA Control and GABBR2 siRNA morphisms in one or more of the rapamycin - sensitive genes alone ; Darker shading = cells treated with additional 100 shown in Table 1 , or one or more polymorphisms in one or ng /ml rapamycin . more genes which affect the expression of one or more of the FIG . 7 Effect of siRNA -mediated down - regulation of rapamycin -sensitive genes shown in Table 1 , wherein the 20 GABBR2 on p - tau expression in SH -SY5Y neuroblastoma presence of at least one variant allele associated with a cells. Grey bars = all single cells ; Vertically -shaded bars =G1 differential response to rapamycin is indicative of the status population ; Horizontally - shaded bars = G2 population ; of mTOR signalling in the cell . Lighter shading = cells treated with Culture medium , siRNA In all aspects of the invention relating to polymorphisms Control and GABBR2 siRNA alone ; Darker shades repre described above , in preferred embodiments , the one or more , 25 sent cells treated with additional 100 ng /ml rapamycin . All two or more , three or more , four or more , five or more , six data are normalised to Control ( 100 % ) . or more , seven or more, eight or more, nine or more , ten or FIG . 8 Effect of siRNA -mediated down - regulation of more polymorphisms for the purposes of genotyping or HOXD10 on cell cycle kinetics in SH -SY5Y neuroblastoma detection are in one or more of the rapamycin -sensitive cells . Vertically - shaded bars = G1 population ; Horizontally genes selected from LILRB2 , FAM5C , CRP , CLU , 30 shaded bars =G2 population ; Lighter shading = cells treated FCGR2A , CD1E , FAM5C , LPL , SYK and CUX1 and / or in with Culture medium , siRNA Control and HOXD10 siRNA one or more of the genes which affect the expression of one alone ; Darker shading = cells treated with additional 100 or more rapamycin - sensitive genes , selected from POU2F1 , ng /ml rapamycin . ADRA1A , PRDM1 and LOXL2 . FIG . 9 Effect of siRNA -mediated down - regulation of In further preferred embodiments , the one or more , two or 35 HOXD10 on p -tau expression in SH -SY5Y neuroblastoma more , three or more, four or more , five or more , six or more , cells . Grey bars = all single cells ; Vertically - shaded bars = G1 seven or more, eight or more , nine or more , ten or more population ; Horizontally -shaded bars = G2 population ; polymorphisms is / are selected from the group consisting of: Lighter shading = cells treated with Culture medium , siRNA ( i) the single polynucleotide polymorphisms: rs798893 , Control and HOXD10 siRNA alone ; Darker shades repre rs725106 , rs1341665 , rs1359059 , rs1532278 , 40 sent cells treated with additional 100 ng /ml rapamycin . All rs 1801274 , rs2036108 , rs811925 , rs883524 , data are normalised to Control (100 % ). rs 1065457, rs1148613 , rs295 , rs290258 , rs365836 and FIG . 10 Effect of siRNA -mediated down -regulation of rs569214 ; and KLF2 on cell cycle kinetics in SH -SY5Y neuroblastoma ( ii ) any polymorphism in linkage disequilibrium with the cells. Vertically - shaded bars = G1 population ; Horizontally single nucleotide polymorphisms of ( i ) . 45 shaded bars = G2 population ; Lighter shading = cells treated with Culture medium , siRNA Control and KLF2 siRNA BRIEF DESCRIPTION OF THE DRAWINGS alone ; Darker shading = cells treated with additional 100 ng/ ml rapamycin . FIG . 1 Cell cycle dependent expression of phosphor- tau FIG . 11 Effect of siRNA -mediated down -regulation of (p - tau ) in SH - SY5Y neuroblastoma cells . White bars = G1; 50 KLF2 on p - tau expression in SH - SY5Y neuroblastoma cells . Black bars = G2 . Grey bars = all single cells ; Vertically - shaded bars =G1 popu FIG . 2 Effect of rapamycin on cell cycle kinetics in lation ; Horizontally -shaded bars = G2 population ; Lighter SH - SY5Y neuroblastoma cells . Vertically - shaded bars = G1 shading = cells treated with Culture medium , siRNA Control population ; Horizontally - shaded bars = G2 population ; and KLF2 siRNA alone ; Darker shades represent cells Lighter shading control cells treated with culture medium ; 55 treated with additional 100 ng / ml rapamycin . All data are Darker shading = cells treated with 100 ng/ ml rapamycin . normalised to Control (100 % ) . FIG . 3 Effect of rapamycin on p - tau expression in FIG . 12 Effect of siRNA -mediated down -regulation of SH -SY5Y neuroblastoma cells . Grey bars = all single cells ; RHO on cell cycle kinetics in SH -SY5Y neuroblastoma Vertically - shaded bars = G1 population ; Horizontally -shaded cells . Vertically - shaded bars = G1 population ; Horizontally bars = G2 population ; Lighter shading = control cells treated 60 shaded bars = G2 population ; Lighter shading = cells treated with culture medium ; Darker shading = cells treated with 100 with Culture medium , siRNA Control and RHO siRNA ng /ml rapamycin . All data are normalised to Control alone ; Darker shading = cells treated with additional 100 ( 100 % ) . ng / ml rapamycin . FIG . 4 Effect of siRNA -mediated down - regulation of FIG . 13 Effect of siRNA -mediated down - regulation of CACNA1 D on cell cycle kinetics in SH - SY5Y neuroblas - 65 RHO on p - tau expression in SH - SY5Y neuroblastoma cells . toma cells . Vertically - shaded bars =G1 population ; Horizon - Grey bars = all single cells ; Vertically -shaded bars = G1 popu tally -shaded bars = G2 population ; Lighter shading = cells lation ; Horizontally -shaded bars = G2 population ; Lighter US 9 ,944 ,986 B2 shading = cells treated with Culture medium , siRNA Control 2 , 3 , 4 and 5 are grouped herein as targets within themTOR and RHO siRNA alone; Darker shades represent cells treated signalling pathway . Tables 2 and 4 show the rapamycin with additional 100 ng/ ml rapamycin . All data are nor - sensitive genes that have an altered expression in the brain malised to Control ( 100 % ) . of Alzheimer ' s patients with mild disease and Tables 3 and FIG . 14 Effect of siRNA -mediated down -regulation of 5 5 show the rapamycin -sensitive genes that have an altered GLI2 on cell cycle kinetics in SH -SY5Y neuroblastoma expression in the brain of Alzheimer ' s patients with cells . Vertically - shaded bars = G1 population ; Horizontally advanced disease . shaded bars = G2 population ; Lighter shading = cells treated Therapeutic Methods with Culture medium , siRNA Control and GLI2 siRNA In a first aspect, the present invention provides methods alone; Darker shading = cells treated with additional 100 10 for the prevention and /or treatment of live human subjects ng /ml rapamycin . diagnosed with Alzheimer ' s disease involving administra FIG . 15 Effect of siRNA -mediated down -regulation of tion of pharmacological agents which modulate or are GLI2 on p -tau expression in SH -SY5Y neuroblastoma cells. capable of modulating one or more , two or more , three or Grey bars = all single cells ; Vertically -shaded bars = G1 popu - more , four or more etc . targets within the mTOR signalling lation ; Horizontally - shaded bars = G2 population ; Lighter 15 pathway . shading = cells treated with Culture medium , siRNA Control In certain embodiments, the therapeutic target is selected and GLI2 siRNA alone ; Darker shades represent cells from the rapamycin - sensitive genes shown in Tables 2 , 3 , 4 treated with additional 100 ng /ml rapamycin . All data are and 5 and the pharmacological agent will typically modulate nonormalised to Control ( 100 % ) . the target via effects at the level of gene expression . In FIG . 16 T , weighted MRI images of ( A ) rapamycin - 20 preferred embodiments, the one or more targets within the treated animals , ( B ) ketamine - treated animals and ( C ) con - mTOR signalling pathway is / are selected from the group of trol animals . rapamycin - sensitive genes consisting of calcium channel , FIG . 17 The effect of mTOR modulation on phosphati voltage - dependent, L type , alpha 1D subunit (CACNA1 D ) , dylcholine species in the brain . gamma - aminobutyric acid B receptor, 2 (GABBR2 ) , FIG . 18 The effect ofmTOR modulation on choline ( A ) 25 homeobox D10 (HOXD10 ) , Kruppel- like factor 2 (KLF2 ) . and creatine ( B ) levels in the brain . rhodopsin (RHO ) and GLI zinc finger family 2 (GLI2 ) . In certain embodiments , the pharmacological agent may DETAILED DESCRIPTION act by up - regulating / increasing or down - regulating / decreas ing expression of the target gene . Gene expression may be The present invention is directed to methods for prevent- 30 detected at the level of the transcriptional product or at the ing , treating and / or diagnosing Alzheimer ' s disease in live level of the protein produced , using standard techniques human subjects involving use of gene targets within the described herein below . Up - regulation or down - regulation “ mTOR signalling pathway " . of gene expression is measured relative to the situation in the The kinase , “mTOR ” , functions within the context of two absence of pharmacological agent, or in the presence of an cytoplasmic protein complexes known as mTORC1 and 35 appropriate , inactive control. mTORC2 . It is however , only themTORC1 complex that is In certain embodiments of the invention , the term “ target" sensitive to the inhibitor rapamycin . Thus, the targets of may be used to refer to the transcriptional products of the interest in the present invention may also be classified as rapamycin - sensitive genes shown in Tables 2 , 3 , 4 and 5 , or targets within the mTORC1 signalling pathway . fragments thereof. The term “ transcriptional product ” is The cytoplasmic kinase mTOR is stimulated or activated 40 intended to encompass the pre- mRNA species generated by a wide variety of upstream signals . These include signals following transcription of the gene , any of the splice inter generated or triggered as a result of nutrient sensing , mediates generated during pre -mRNA processing and the hypoxia , and / or the activity of growth factors and their mature fully -spliced mRNA species. Inhibition of such cognate receptors. Activation of mTOR upregulates its transcriptional products may involve down - regulating the kinase activity and thereby increases mTOR -mediated phos - 45 level of such products , for example , by promoting nucleic phorylation of downstream protein targets within the cell. In acid degradation . This may be mediated for example , by most cases, the direct downstream protein targets of mTOR interfering RNA species , such as siRNAs . interact with a variety of further molecular targets , and in In certain other embodiments of the invention , the " tar doing so , stimulate a wide variety of cellular responses , such get” may refer to the proteins encoded by the rapamycin as increased protein synthesis and the promotion of cell 50 sensitive genes shown in Tables 2 , 3 , 4 and 5 , or fragments growth and proliferation . The chain of molecular events thereof. The pharmacological agent may therefore bring triggered downstream of mTOR -mediated phosphorylation about an increase or decrease in the level of protein within of its direct protein targets is defined herein as the “ mTOR a cell or an increase or decrease in the biological activity of signalling pathway " , and the target genes /proteins of the the protein . present invention fall within this pathway . 55 As noted above , the target of the invention may include a In the methods of the present invention , the particular fragment of the transcriptional product of the rapamycin targets of interest within the mTOR signalling pathway are sensitive genes shown in Tables 2 , 3 , 4 and 5 or a fragment selected from the genes shown in Tables 2 , 3 , 4 and 5 . The of a protein encoded by one or more of the rapamycin term “ target ” is intended to encompass the genes of Tables sensitive genes shown in Tables 2 , 3 , 4 and 5 . The term 2 , 3 , 4 and 5 , the transcriptional products of such genes and 60 " fragment” should be taken to mean a form of the transcrip the proteins encoded by such genes . tional product or protein , which is reduced in length by one The particular genes shown in Table 1 are “ rapamycin - or more , two or more, three or more , four or more etc. sensitive ” for the reason that their cellular expression is nucleotides or amino acids, respectively , as compared with affected ( increased or decreased ) by the compound rapamy - the full- length transcriptional product or protein . In relation cin . Since rapamycin is known to inhibit the serine /threonine 65 to post -transcriptional products , the term “ fragment" may kinase mTOR in (human ) cells , and thereby reduce signal- also be applied to alternatively - spliced forms of mRNA ling downstream of this protein , the genes shown in Tables produced from the originating pre -mRNA transcript. US 9 ,944 ,986 B2 10 In the context of the present invention , the term “ modu Aerosol preparations suitable for inhalation may include lates ” is used very broadly to mean an agent capable of solutions and solids in powder form , which may be com changing or altering the expression of a gene , the production bined with a pharmaceutically acceptable carrier , such as an and /or level of a transcriptional product of a gene and /or the inert compressed gas. production , level and /or activity of a protein encoded by a 5 Also encompassed are dosage forms for transdermal gene. The term “ modulates” may be used to describe an administration , including creams, lotions , aerosols and /or emulsions . These dosage forms may be included in trans increase or a decrease in any of the parameters described dermal patches of the matrix or reservoir type , which are above . Any increase or decrease is measured relative to the generally known in the art . situation present in the absence of the pharmacological agent** * 10 Pharmaceutical preparations may be conveniently pre or in the presence of a suitable inactive control. pared in unit dosage form , according to standard procedures In one embodiment, the pharmacological agent is an of pharmaceutical formulation . The quantity of active com activator or agonist capable of increasing or up - regulating pound per unit dose may be varied according to the nature the expression of a gene , the production and / or level of a of the active compound and the intended dosage regime. transcriptional product of a gene and / or the production , level 15 Generally this will be within the range 0 . 1 mg to 1000 mg. and/ or activity of a protein encoded by a gene . In an Wherein the pharmacological agent consists of a nucleic alternative embodiment, the pharmacological agent is an acid therapeutic agent , for example , an antisenseantisense RNA inhibitor or antagonist capable of decreasing or down species or a double - stranded RNA species for use as an RNA regulating the expression of a gene , the production and / or interfering agent, the active agent may be administered to a level of a transcriptional product of a gene and/ or the 20 patient in need thereof via gene therapy approaches . production , level and /or activity of a protein encoded by a The rapamycin - sensitive target genes shown in Tables 2 , gene . 3 , 4 and 5 encode a range of proteins including enzymes , Classes of pharmacological agents suitable for use in receptors and transporters. Preferred known pharmacologi accordance with the methods described herein would be cal agents for use in conjunction with the present methods available to those skilled in the art. Such agents include but 25 include the following ; however, this is not to be construed as are not limited to small molecules, organic or inorganic limiting the invention to these specific embodiments. molecules , biological molecules including antibodies and wherein the target gene is CACNA1 D , pharmacological antigen binding fragments thereof, natural or synthetic poly agents for use may include MEM - 1003, clevidipine peptides or peptides , nucleic acid therapeutic agents includ - butyrate , aliskiren / amlodipine /hydrochlorothiazide , mibe ing antisense RNA species and double - stranded RNA spe - 30 fradil, bepridil , nisoldipine , isradipine, amlodipine and /or cies for use as RNA interfering agents , for example siRNA nicardipine. molecules . Wherein the target gene is HDAC5 , pharmacological In the context of the present invention , the term antibody agents for use may include tributyrin , belinostat, pyroxam covers native immunoglobulins from any species, chimeric ide, vorinostat and /or romidepsin . antibodies , humanised antibodies, F (ab ') 2 fragments , Fab 35 Wherein the target gene is IL6 , pharmacological agents fragments , Fv fragments , sFv fragments and highly related for use may include tocilizumab . molecules such as those based upon antibody domains Wherein the target gene is NR3C1, pharmacological which retain specific binding affinity ( for example , single agents for use may include rimexolone , medrysone , clocor domain antibodies ). tolone pivalate , diflorasone diacetate , fluorometholone , dex Pharmacological agents may be formulated as composi - 40 amethasone phosphate, cortisone acetate , halcinonide , flu tions for delivery wherein the agent in a suitable dosage randrenolide , desoximetasone , desonide , prednisolone , form is combined with a pharmaceutically acceptable carrier clobetasol propionate , fluocinolone acetonide , prednisone , such as a diluent, filler , salt, buffer, stabilizer , solubilizer etc . hydrocortisone, triamcinolone , dexamethasone 21 -acetate , The dosage form may contain other pharmaceutically 1 1beta hydrocortisone acetate , betamethasone , dexametha acceptable excipients for modifying conditions such as pH , 45 sone , budesonide , fluticasone, beclomethasone dipropi osmolarity , taste , viscosity , sterility , lipophilicity , solubility onate , acetic acid /hydrocortisone , betamethasone acetatel etc . betamethasone phosphate , betamethasone acetate , Suitable dosage forms include solid dosage forms, for triamcinolone acetonide, ciprofloxacin /hydrocortisone , dex example , tablets , capsules, powders, dispersible granules , amethasone/ neomycin /polymyxin B , ciprofloxacin /dexam cachets and suppositories , including sustained release and 50 ethasone , ORG 34517 , ciclesonide , betamethasone dipropi delayed release formulations . Powders and tablets will gen - onate /calcipotriene , fluticasone furoate , budesonidel erally comprise from about 5 % to about 70 % active ingre - formoterol, difluprednate , formoterol/ mometasone furoate , dient. Suitable solid carriers and excipients are generally clotrimazole /betamethasone dipropionate , fluticasone/ sal known in the art and include, e . g . magnesium carbonate , meterol, dexamethasone / tobramycin , clotrimazole /betame magnesium stearate , talc , sugar, lactose , etc . Tablets , pow - 55 thasone, miconazole , prednisolone acetate , clioquinol/ hy ders , cachets and capsules are all suitable dosage forms for drocortisone , methylprednisolone acetate , mometasone oral administration . furoate , amcinonide , methylprednisolone succinate , betame Liquid dosage forms include solutions , suspensions and thasone phosphate , fluocinonide, prednicarbate , hydrocorti emulsions . Liquid form preparations may be administered sone cypionate , hydrocortisone succinate, prednisolone by intravenous, intracerebral, intraperitoneal, parenteral or 60 phosphate , betamethasone valerate , betamethasone benzo intramuscular injection or infusion . Sterile injectable formu - ate , fludrocortisone acetate , prednisolone tebutate , betame lations may comprise a sterile solution or suspension of the thasone dipropionate , hydrocortisone buteprate , alclometa active agent in a non - toxic , pharmaceutically acceptable sone dipropionate , hydrocortisone butyrate , diluent or solvent. Suitable diluents and solvents include fluorometholone acetate , hydrocortisone valerate , nystatin / sterile water , Ringer' s solution and isotonic sodium chloride 65 triamcinolone acetonide , loteprednol etabonate , hydrocorti solution , etc . Liquid dosage forms also include solutions or sone phosphate , methylprednisolone , halobetasol propi sprays for intranasal administration . onate , flunisolide and /or mifepristone . US 9 ,944 , 986 B2 Wherein the target gene is NTSR1, pharmacological The human subject to be treated according to the methods agents for use may include contulakin - G . provided herein may be any human subject diagnosed as Wherein the target gene is PRKCH , pharmacological having Alzheimer 's disease . This includes individuals with agents for use may include ingenol 3 - angelate . early -onset familial Alzheimer ' s disease and individuals Wherein the target gene is SCN8A , pharmacological 5 with late -onset sporadic forms of this disease . agents for use may include riluzole . Screening Methods Wherein the target gene is SERPINE1 , pharmacological In a second aspect, the present invention also provides agents for use may include drotrecogin alfa . methods of screening for pharmacological agents useful in Wherein the target gene is TRPV1, pharmacological the prevention and / or treatment of Alzheimer ' s disease in a agents for use may include SB -705498 , resiniferatoxin and1 / 10 subject, wherein said method comprises the steps of: or capsaicin . ( i ) contacting a cell with a test pharmacological agent; ( ii ) measuring either the expression level of one or more Wherein the target gene is VEGFA , pharmacological of the rapamycin - sensitive genes shown in Tables 2 , 3 , agents for use may include bevacizumab , ranibizumab , 4 and 5 , or the level or activity of one or more of the aflibercept and /or pegaptanib . 15 proteins encoded by the rapamycin - sensitive genes Wherein the target gene is GLP1 R , pharmacological shown in Tables 2 , 3 , 4 and 5 ; agents for use may include liraglutide, T - 0632 , GLP - 1 ( 7 - 36 ) ( iii ) measuring either the expression level of one or more amide and / or exenatide . of the rapamycin -sensitive genes shown in Tables 2 , 3 , The term “ Alzheimer' s disease” is used herein broadly to 4 and 5 , or the level or activity of one or more of the mean disease diagnosed on the basis of clinical criteria 20 proteins encoded by the rapamycin - sensitive genes and /or disease identified on the basis of pathophysiological shown in Tables 2 , 3 , 4 and 5 in a control cell not changes associated with AD . exposed to the test pharmacological agent ; At present, the diagnosis of Alzheimer ' s disease in live ( iv ) comparing the results determined in steps ( ii ) and ( iii ) human subjects is based on the evaluation of clinical criteria , wherein a difference in the expression level of one or such as the NINCDS/ ADRDA criteria (McKhann , G . et al. , 25 more of the rapamycin - sensitive genes shown in Tables ( 1984 ) Neurology 34 : 939 - 944 ) . However, such diagnostic 2 , 3 , 4 and 5 , or the level or activity of one or more of criteria applied in the clinic are based on the measurement the proteins encoded by the rapamycin -sensitive genes of cognitive parameters , and are thus reliant on the onset of shown in Tables 2 , 3 , 4 and 5 , indicates that the test cognitive symptoms in patients with this disease . pharmacological agent is suitable for use in the pre It is however , clear from extensive research carried out 30 vention and /or treatment of Alzheimer ' s disease. that the pathophysiological or changes defining AD are In preferred embodiments , the one or more targets within detectable in individuals years before these patients show the mTOR signalling pathway is / are selected from the group any signs of cognitive impairment. Alzheimer ' s disease has of rapamycin -sensitive genes consisting of calcium channel, accordingly , been classified into three phases : voltage -dependent , L type, alpha 1D subunit ( CACNA1 D ) , (i ) asymptomatic Alzheimer ' s disease (preclinical stage ); 35 gamma - aminobutyric acid B receptor, 2 (GABBR2 ) , ( ii ) mild cognitive impairment (MCI ) due to Alzheimer ' s homeobox D10 (HOXD10 ), Kruppel- like factor 2 (KLF2 ) , disease (pre -dementia stage ) ; and rhodopsin ( RHO ) and GLI zinc finger family 2 (GLI2 ) . ( iii ) clinically - defined Alzheimer ' s disease ( dementia ). The pharmacological agents for testing in the screening In the context of the present invention , the phrase " pre - methods provided herein may be selected from any class of vention and /or treatment of Alzheimer ' s disease ” is intended 40 agent as described in the context of the therapeutic methods to encompass prevention and / or treatment strategies used for of the present invention . The methods may involve screen an individual having disease at any one of the three phases ing one or more pharmacological agents simultaneously , for defined above . example in a multiplex format. Agents for use in the It is not at present possible to reliably diagnose individu screening methods may be provided in any suitable format, als with asymptomatic AD or early -stage disease ; however , 45 including compound libraries . as methods of diagnosis improve , it may prove possible to The “ difference ” in gene expression and / or protein activ identify individuals with neuropathological changes defin - ity to be detected using the screening method described ing the early stages of AD . The methods described herein herein may be established according to the sensitivity may therefore be used to prevent and/ or delay the onset of requirements of those using the method to identify pharma cognitive symptoms in a subject asymptomatic for Alzheim - 50 cological agents suitable for the prevention and / or treatment er ' s disease . This may be achieved by a reversal , stabilisa - of AD . tion and/ or delay of the neurological changes underlying AD In certain embodiments , the difference may be measured pathology . as a decrease in the expression of one or more of the Themethods provided herein may also be used to prevent rapamycin -sensitive genes shown in Tables 2 , 3 , 4 and 5 as and / or delay the onset of AD -associated dementia in indi- 55 compared with control cells not exposed to the pharmaco viduals who are already symptomatic to varying degrees . logical agent , wherein said decrease indicates that the test For example , the methods of the invention may be applied pharmacological agent is suitable for use in the prevention to individuals classified according to standard criteria , for and / or treatment of Alzheimer ' s disease . example the Mayo Clinic diagnostic criteria (Winblad et al. , Any difference or decrease in gene expression may be ( 2004 ) J . Intern . Med 256 : 240 - 246 ) , as having mild cog - 60 measured by assessing the level of transcriptional product or nitive impairment (MCI ) . In a further embodiment, the mRNA produced from any of the rapamycin - sensitive genes method of the invention may be used to prevent and /or delay shown in Tables 2 , 3 , 4 and 5 . Alternatively or in addition , the worsening of symptoms in individuals already diagnosed any difference or decrease may be measured by assessing the with clinical dementia . Themethods of the present invention levels of protein produced from any of the rapamycin may therefore be used to treat Alzheimer ' s disease in a 65 sensitive genes shown in Tables 2 , 3 , 4 and 5 . subject exhibiting mild cognitive impairment or in a subject Suitable methods for the detection / quantitation of tran exhibiting clinical dementia . scriptional products which may be used in accordance with US 9 ,944 ,986 B2 13 14 the present methods are well known in the art , and include , classified as “ possible Alzheimer ' s disease ” or “ probable but are not limited to hybridisation techniques , such as Alzheimer ' s disease ” based on the results of current diag Northern blotting or microarray technologies , and amplifi- nostic tests . cation - based techniques such as RT- PCR or nucleic -acid The presentmethods may therefore be used to " assist with sequence- based amplification (NASBA ). 5 diagnosis ” meaning that they are used to assess the likeli Suitable techniques for assessing protein levels are known hood that an individual has Alzheimer ' s disease at any one in the art and include , but are not limited to , flow cytometry, of the three phases of the disease described above . In immunoblot analysis , ELISA , Elispot and Fluorospot preferred embodiments , the presentmethod may be used to assays . In certain embodiments , these assays may be used in assist with diagnosis of early - stage Alzheimer 's disease in conjunction with commercially - available antibodies that 10 asymptomatic patients or patients exhibiting mild cognitive bind to the protein of interest, in order to determine protein impairment. levels . Standard assays are also available for measuring the The methods of the invention may also be used in activity of certain proteins , for example standard enzyme combination or together with other methods or tests used for activity assays , such as kinase assays . Alzheimer ' s disease diagnosis , for example in order to Diagnostic Methods 15 improve the specificity and /or sensitivity of these methods In a further aspect, the current invention provides a or tests . In specific embodiments , the present methods may method to assist with diagnosis of Alzheimer ' s disease in a be carried out in combination with a test designed to monitor live human subject. one or more biomarkers of Alzheimer 's disease in a par In the context of the present invention , the term “ diag . ticular individual , and the combined result may be used to nosis of Alzheimer ' s disease ” is used very broadly and 20 assess the likelihood that the individual has Alzheimer ' s should be taken to mean diagnosis of an individual having disease . In alternative embodiments , the present method disease at any one of the three phases of the disease defined may be used to independently substantiate the results of above i . e . the preclinical stage , the pre - dementia stage or the other diagnostic tests . dementia stage . In one embodiment, the method of the The present method is intended to provide a means to invention is used to diagnose or assist with diagnosis of 25 diagnose and / or assist with diagnosis of Alzheimer ' s disease Alzheimer ' s disease in its preclinical stage in an individual in multiple settings . In one embodiment, the present method with no symptoms of disease , for example no signs of may be used to diagnose individuals with Alzheimer ' s cognitive impairment. In other embodiments , the same basic disease so as to identify patients suitable for the assessment methodology may be used to screen subjects who are of new Alzheimer ' s disease treatments , for example the “ symptomatic ” to varying degrees. For example , the method 30 identification of suitable subjects for clinical trials . New of the invention may be applied to individuals classified treatments or therapies designed to be preventive and / or according to standard criteria , for example the Mayo Clinic curative may only have the best chance of success in patients diagnostic criteria (Winblad et al. , (2004 ) J . Intern . Med 256 : with asymptomatic or early - stage disease. The present 240 - 246 ) , as having mild cognitive impairment (MCI ) . Not method may therefore be used to diagnose or assist with all patients classified as having MCI will have the type of 35 diagnosis of pre -clinical Alzheimer ' s disease in asymptom underlying neurodegeneration associated with Alzheimer ' s atic individuals or to diagnose or assist with diagnosis of disease . Thus , the presentmethod may be used to distinguish individuals with MCI that have underlying Alzheimer ' s or assist with distinguishing between individuals with MCI disease pathology, for the purposes of assessing new treat that have underlying Alzheimer ' s disease and therefore are ments specifically in these patients . As improved treatments likely to go on to develop Alzheimer ' s disease - associated 40 for Alzheimer ' s disease become available , the present meth dementia , and those that have MCI attributable to a different ods may also be used to diagnose or assist with diagnosis of cause or condition . In a further embodiment of the invention , individuals so as to identify patients who will benefit from the method may be used to diagnose or assist with diagnosis treatments that may have the ability to prevent cognitive of Alzheimer' s disease in a human subject exhibiting one or decline . more symptoms consistent with Alzheimer' s disease . 45 The present invention also provides methods of assessing The diagnostic methods of the present invention may also the risk of Alzheimer ' s disease progression in a human be used in conjunction with existing diagnostic criteria , for subject. In this context, “ Alzheimer ' s disease progression ” example the NINCDS /ADRDA criteria , in order to verify or should be taken to mean the progressive neurodegeneration substantiate an Alzheimer' s disease diagnosis in a human associated with this disease and/ or the progressive decline in subject who already meets the existing criteria for a positive 50 cognitive function that accompanies the underlying neuro diagnosis . In this embodiment, the present methods may pathology . Such methods may be applied to individuals provide an adjunct to alternative diagnostic tests , wherein suspected of having any one of the three phases of Alzheim the present methods are independent of neuropsychological er ' s disease defined above , or individuals considered at risk symptoms. This may allow for a more reliable diagnosis of of developing this disease . clinical Alzheimer ' s disease , particularly since not all 55 In preferred embodiments of the invention , the methods patients presenting with dementia symptoms will have provide means by which to assess or predict cognitive Alzheimer 's disease as the underlying cause . decline in human subjects by identifying individuals with The presentmethods are used in particular, to assist with early - stage Alzheimer ' s disease who will go on to develop diagnosis of Alzheimer 's disease in a live human subject. A Alzheimer ' s disease -associated dementia . In certain definitive diagnosis of Alzheimer ' s disease is generally 60 embodiments , the individual or human subject for testing considered by those in the field to be impossible in a live will be asymptomatic for Alzheimer ' s disease . In alternative subject, and can only be made post -mortem following patho - embodiments , the individual or human subject for testing logical examination of brain tissue from the patient. Thus , will exhibit mild cognitive impairment or will exhibit one or although present methods may seek to “ diagnose ” Alzheim - more symptoms consistent with Alzheimer ' s disease . er ' s disease in live subjects , such a diagnosis is typically 65 In the diagnostic methods and the methods of assessing based on an assessment of the likelihood that any given the risk of disease progression described above , the method individual has the disease . In this regard, individuals may be comprises as a first step measuring either the expression US 9 ,944 ,986 B2 15 16 level of one or more of the rapamycin - sensitive genes shown Methods of Assessing Pre -disposition to Alzheimer ' s Dis in Tables 2 , 3 , 4 and 5 and / or the activity of one or more of ease the proteins encoded by the rapamycin - sensitive genes In a further aspect , the current invention also provides shown in Tables 2 , 3 , 4 and 5 . Gene expression may be methods for screening a human subject for pre -disposition to analysed by assessing levels ofmRNA produced following 5 Alzheimer ' s disease , wherein said method comprises the gene transcription or by assessing levels of the protein steps of: produced following translation of the mRNA . The detection ( i) measuring either the expression level of one or more of ofmRNA and protein levels may be carried out by methods the rapamycin -sensitive genes shown in Tables 2 , 3 , 4 known to those skilled in the art. Protein activity may also and 5 , and / or the activity of one or more of the proteins be analysed using suitable assays known to those skilled in encoded by the rapamycin -sensitive genes shown in the art . Tables 2 , 3 , 4 and 5 ; In certain embodiments of the invention , protein activity ( ii ) comparing the expression level and /or activity mea may be measured directly . For example , the activity of a sured in ( i ) with reference /control values , kinase enzyme may be measured using an assay that detects 15 wherein a difference in expression level and / or activity of phosphorylation of the enzyme' s direct substrate . Protein the one or more rapamycin sensitive genes shown in activity may also be measured indirectly by measuring Tables 2 , 3 , 4 and 5 is taken to mean the subject is alterations and /or changes in the level and / or activity of pre - disposed to Alzheimer' s disease . metabolites linked to the activity of the proteins encoded by In certain embodiments of the invention , the difference the rapamycin - sensitive genes shown in Tables 2 , 3 , 4 and 5 . 20 may be measured as an increase in the expression level of one or more of the rapamycin - sensitive genes shown in As a second step , the methods require a comparison Tables 2 , 3 , 4 and 5 , and / or the activity of one or more of the between the gene expression levels and /or protein activity proteins encoded by the rapamycin -sensitive genes shown in measured in the first step and the gene expression levels Tables 2 , 3 , 4 and 5 as compared with reference /control and / or protein activity defined as reference or control values. 25 values and such increase is taken to mean the subject is Such reference or control values may be determined from pre - disposed to Alzheimer 's disease . Such “ pre - disposition measurements made using age -matched healthy subjects , to Alzheimer' s disease ” may be manifest as an increased preferably wherein such subjects exhibit no signs of cogni- lifetime risk of developing Alzhimer ' s disease as compared tive impairment. If the method is carried out with the with the average lifetime risk associated with the general intention of assessing the risk of Alzheimer 's disease pro - 30 population , and /or as an earlier age of onset of Alzheimer' s gression , the reference or control values may consist of disease in affected individuals . values determined from measurements made using the same As described above in connection with the diagnostic human subject at an earlier point in time, for example 1 , 2 , methods aspect of the invention , the presently - claimed 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 or 11 months earlier, or 1 , 2 , 3 , 4 , 5 , 6 , methods comprise as a first step measuring either the expres 7 , 8 , 9 , 10 etc years earlier. A comparison between mea - 35 sion level of one or more of the rapamycin - sensitive genes surements determined for the same human subject at differ shown in Tables 2 , 3 , 4 and 5 and / or the activity of one or ent points in timemay assist in determining whether there more of the proteins encoded by the rapamycin - sensitive genes shown in Tables 2 , 3 , 4 and 5 . Gene expression may has been a change in the level of expression of a gene and / or be analysed by assessing levels of mRNA produced follow activity of a protein over time that is indicative of Alzheim 40 ing gene transcription or by assessing levels of the protein er 's disease progression . produced following translation of the mRNA . The detection The genes and /or proteins for use in conjunction with the ofmRNA and protein levels may be carried out by methods diagnostic methods of the present invention are selected known to those skilled in the art . Protein activity may also from the rapamycin - sensitive genes shown in Tables 2 , 3 , 4 be analysed using suitable assays known to those skilled in and 5 . In preferred embodiments , the one or more targets 45 the art . within the mTOR signalling pathway is / are selected from In certain embodiments of the invention , protein activity the group of rapamycin -sensitive genes consisting of cal - may be measured directly . For example , the activity of a cium channel , voltage -dependent , L type , alpha 1D subunit kinase enzymemay be measured using an assay that detects (CACNA1D ), gamma - aminobutyric acid B receptor, 2 phosphorylation of the enzyme' s direct substrate . Protein (GABBR2 ) , homeobox D10 (HOXD10 ) , Kruppel -like fac - 50 activity may also be measured indirectly by measuring tor 2 (KLF2 ) , rhodopsin (RHO ) and GLI zinc finger family alterations and/ or changes in the level and/ or activity of metabolites linked to the activity of the proteins encoded by 2 (GLI2 ) . the rapamycin - sensitive genes shown in Tables 2, 3, 4 and 5 . The measurement of gene expression and/ or protein activ As a second step , the methods require a comparison ity may be made using any suitable sample taken from the 55 between the gene expression levels and /or protein activity subject. In a preferred embodiment, the sample is derived measured in the first step and the gene expression levels from the cerebrospinal fluid of a patient. In certain embodidin and / or protein activity defined as reference or control values . ments , the level or gene expression and / or protein activity Such reference or control values may be determined from may be determined using imaging techniques, preferably measurements made using age -matched healthy subjects , non - invasive imaging of the subject' s brain or regions 60 preferably wherein such subjects exhibit no signs of cogni thereof. In certain embodiments , imaging techniques may be tive impairment. Alternatively or in addition , such reference used to detect alterations and /or temporal changes in brain or control values may be determined from measurements metabolites , such as choline or creatine, that are indicative made using subjects that are known not to have developed of a change in protein activity. Imaging techniques thatmay Alzheimer ' s disease during their lifetime. be particularly useful in conjunction with the present meth - 65 The screening methods described herein will typically be ods include but are not limited to PET, SPECT, MR spec - used to assess the pre -disposition to Alzheimer ' s disease in troscopy and functional MRI. individuals that are otherwise asymptomatic for this disease . US 9 ,944 ,986 B2 17 18 Methods of Assessing Pre - disposition to Alzheimer ' s Dis - polymorphic variants in genes which affect the expression of ease Based on Detection of Polymorphisms rapamycin -sensitive genesmay be located in genes encoding In a further aspect, provided herein is a method of regulators , particularly upstream regulators , of expression of screening a human subject for pre -disposition to Alzheim genes in the mTOR signalling pathway described herein . er ' s disease , which method comprises genotyping the sub - 5 In the context of the present invention , a polymorphism ject for a polymorphism in one or more of the rapamycin - “ in ” a gene should be taken to mean a genetic variant present sensitive genes shown in Table 1 , wherein the presence of at at any position within the full -length native gene . Polymor least one variant allele associated with Alzheimer ' s disease phisms may therefore be located in exons , introns or in is an indication that the subject is pre - disposed to Alzheim - regulatory regions located upstream or downstream of the er ' s disease . 10 coding segment. In this aspect of the invention , " a polymorphism ” can be In preferred embodiments , the method comprises geno taken to mean one or more polymorphisms. Therefore , typing a subject for one or more polymorphisms in one or provided herein is a method of screening a human subject for more rapamycin - sensitive genes selected from LILRB2, pre -disposition to Alzheimer' s disease , which method com - FAM5C , CRP, CLU , FCGR2A , CD1E , FAM5C , LPL , SYK prises genotyping the subject for one or more polymor - 15 and CUX1. Alternatively , or in addition , the method may phisms in one or more of the rapamycin - sensitive genes comprise genotyping a subject for one or more polymor shown in Table 1 , wherein the presence of at least one phisms in one or more genes which affect the expression of variant allele associated with Alzheimer ' s disease is an one or more rapamycin - sensitive genes , wherein the “ regu indication that the subject is pre -disposed to Alzheimer' s latory ” genes are selected from POU2F1 , ADRA1A , disease . 20 PRDM1 and LOXL2 . In a still further aspect , provided herein is a method of POU2F1 is a transcriptional regulator of the mTOR screening a human subject for pre- disposition to Alzheim - genes : A2M , CRP , CSF1R , CYP2C9 , ESR1, GSTM3, IL2 , er' s disease , which method comprises genotyping the sub IL6 , PRKAA2 , SPP1, TLR4 from Table 1 . ADRA1A is a ject for one or more polymorphisms in one or more genes regulator of mTOR regulated genes: CDKN1B , EGR1, which affect the expression of one ormore of the rapamycin - 25 FGF7, FN1, IL6 , JUN , LOX , NR4A1, NR4A2 from Table 1. sensitive genes shown in Table 1 , wherein the presence of at LOXL2 is an upstream regulator of mTOR regulated genes : least one variant allele associated with Alzheimer ' s disease CDH1, FN1, MMP9 from Table 1 . PRDM1 is the upstream is an indication that the subject is pre - disposed to Alzheim - regulator of mTOR regulated genes : ESR1, IGHG1, IL10 , er ' s disease . IL2 , IL6 , MYC , RELN , SCGN from Table 1 . As used herein , the term “ polymorphism ” includes single 30 In preferred embodiments , the screening methods involve nucleotide polymorphisms or SNPs , which are changes in genotyping a subject for one or more , two or more , three or which a single base in the DNA differs from the usual base more , four or more , five or more , six or more , seven or more , at that position . Millions of SNPs have been catalogued eight or more , nine or more , ten or more polymorphisms throughout the human genome, and many of these have been selected from the group of single nucleotide polymorphisms linked to increased or decreased susceptibility or pre - dispo - 35 consisting of rs798893 , rs725106 , rs1341665 , rs1359059 , sition to certain diseases. SNPs found within the ApoE gene rs1532278 , rs1801274 , rs2036108 , rs811925 , rs883524 , have already been linked to elevated risk of Alzheimer ' s rs 1065457 , rs1148613 , rs295 , rs290258 , rs365836 and disease . rs569214 , as characterised in Table 14 . The presence of at Associations between polymorphisms or polymorphic least one variant allele associated with Alzheimer' s disease variants and susceptibility to Alzheimer ' s disease can be 40 is an indication that the subject is pre - disposed to Alzheim identified or confirmed by carrying out genetic association er ' s disease . The bases of the variant alleles associated with studies , for example family - based or case - control associa - Alzheimer ' s disease for the SNPs described above are as tion studies . Associations may also be determined by evalu - follows: rs798893 (C ), rs725106 (A ), rs1341665 ( A ), ating the relationship between deregulated gene expression rs1359059 ( A ) , rs1532278 ( C ) , rs1801274 ( G ) , rs2036108 seen in the brain of Alzheimer ' s disease patients and the 45 ( T ) , rs811925 (G ), rs883524 ( C ) , rs1065457 (G ), rs1148613 underlying genotype . ( C ) , rs295 ( C ) , rs290258 ( G ), rs365836 (G ) and rs569214 In the presentmethods for screening for pre- disposition to ( T ) ( see Table 14 ) . Alzheimer ' s disease , a subject is genotyped for one or more The screening methods may also involve genotyping a polymorphisms or polymorphic variants in one or more of subject for one or more , two or more , three or more , four or the rapamycin - sensitive genes shown in Table 1 . In certain 50 more , five or more , six or more , seven or more , eight or embodiments , the subject may be genotyped for two or more , nine or more , ten or more polymorphisms selected more , three or more , four or more , five or more, six ormore , from the group of polymorphisms consisting of polymor seven or more , eight or more , nine or more , ten or more phisms, particularly SNPs, associated with or in linkage polymorphisms or polymorphic variants in one or more of disequilibrium (LD ) with the single nucleotide polymor the rapamycin - sensitive genes shown in Table 1 . Wherein 55 phisms: rs798893 , rs725106 , rs1341665 , rs1359059 , the methods involve genotyping for more than one poly rs1532278 , rs1801274 , rs2036108 , rs811925 , rs883524 , morphism , the polymorphisms may be in the same rapamy rs1065457 , rs1148613 , rs295 , rs290258 , rs365836 and cin -sensitive gene or in different rapamycin - sensitive genes . rs569214 . Polymorphisms “ associated ” with the SNPs char Alternatively , or in addition , the subject may be genoy - acterised in Table 14 include polymorphisms in close prox typed for one or more , two or more , three or more , four or 60 imity to the identified SNPs . Polymorphisms in linkage more , five or more , six or more , seven or more, eight or disequilibrium with the characterised SNPs could be iden more , nine or more , ten or more polymorphisms or poly tified by one of skill in the art using standard association morphic variants in one or more genes which affect the mapping techniques described in the art . expression of one or more of the rapamycin - sensitive genes The screening methods of the present invention may be shown in Table 1 . Wherein the methods involve genotyping 65 carried out in conjunction with other screening methods for more than one polymorphism , the polymorphisms may used to assess pre - disposition to Alzheimer' s disease includ be in the same gene or in different genes. Polymorphisms or ing use of screening methods based on measuring the US 9 ,944 ,986 B2 20 expression of the rapamycin - sensitive gene targets described tion within a single gene, or across multiple genes, and the elsewhere herein ( see Tables 2 - 5 ) . Other methods for assess accumulated variants may have an additive effect . In view of ing a subject ' s pre- disposition or risk of Alzheimer ' s disease the foregoing, it is within the scope of the invention to may utilise other known risk factors , including in particular perform genotyping of polymorphisms or polymorphic vari environmental risk factors such as plasma homocysteine 5 ants within multiple genes , wherein at least one of the genes levels. is selected from the rapamycin - sensitive genes shown in In accordance with the invention , genotyping ofpolymor Table 1. Such a “ panel screen ” of multiple genes may be phic variants can be carried out using any suitable method used to simultaneously analyse multiple polymorphisms that ology known in the art and it is to be understood that the serve as markers of susceptibility /pre - disposition to invention is in no way limited by the precise technique used 10 Alzheimer 's disease in the same human subject . In a pre to carry out the genotyping . ferred embodiment, genotyping of multiple polymorphisms Known techniques which may be used for genotyping in a single patient sample may be carried out simultaneously , single nucleotide polymorphisms include ligation detection for example with the use of a microarray or “ gene chip ” . reaction (LDR ; Day , D . J. , Speiser, P . W . , White , P . C . & In certain embodiments of the invention , the screening for Barany , F . Genomics 29 , 152 62 ( 1995 ) ) , mass spectrometry , 15 pre -disposition to Alzheimer ' s disease will involve geno particularly matrix - assisted laser desorption / ionization time- typing a subject for multiple polymorphismsor polymorphic of- flight mass spectrometry (MALDI - TOF -MS ), single variants in one or more of the genes shown in Table 1 or nucleotide primer extension and DNA chips or microarrays multiple polymorphisms or polymorphic variants in one or ( see review by Schafer , A . J . and Hawkins, J . R . in Nature more genes which affect the expression of one or more of the Biotechnology , Vol 16 , pp33 - 39 ( 1998 ) . The use of DNA 20 rapamycin - sensitive genes shown in Table 1 , or a combina chips or microarrays may enable simultaneous genotyping at tion of both . In the context of the present invention , “ mul many different polymorphic loci in a single individual or the tiple ” should be taken to mean two or more , three or more, simultaneous genotyping of a single polymorphic locus in four or more , five or more, six or more etc . The presence of multiple individuals . SNPs may also be scored by DNA multiple variant alleles associated with Alzheimer ' s disease sequencing. 25 is an indication that the subject is pre - disposed to Alzheim In addition to the above , SNPs are commonly scored er ' s disease . using PCR -based techniques , such as PCR - SSP using allele - In further preferred embodiments , the screening methods specific primers (described by Bunce M , et al. , Tissue will involve genotyping a subject for a combination of single Antigens , 1995 ; 50 : 23 - 31 ) . This method generally involves polynucleotide polymorphisms selected from the following : performing DNA amplification reactions using genomic 30 ( i) rs1065457 , rs798893 and rs2036108 ; DNA as the template and two different primer pairs , the first ( ii ) rs1065457 , rs1148613, rs290258 , rs725106 and primer pair comprising an allele -specific primer which under rs 1341665 ; appropriate conditions is capable of hybridising selectively ( iii) rs1065457, rs295 , rs1359059, rs1532278 , rs798893 , to the wild type allele and a non allele - specific primer which rs365836 , rs725106 and rs1341665; binds to a complementary sequence elsewhere within the 35 ( iv ) rs1148613 , rs290258 , rs725106 , rs1341665 and gene in question , the second primer pair comprising an rs2036108 ; allele- specific primer which under appropriate conditions is ( v ) rs 1065457 , rs1148613 , rs290258 , rs725106 , capable of hybridising selectively to the variant allele and rs 1341665 and rs2036108 ; the same non allele -specific primer. Further suitable tech (vi ) rs1065457 , rs1359059 , rs725106 and rs2036108 ; or niques for scoring SNPs include PCR ELISA and denaturing 40 ( vii ) rs798893 and rs725106 , high performance liquid chromatography (DHPLC ) . wherein the presence of a combination of variant alleles If the SNP results in the abolition or creation of a associated with Alzheimer ' s disease is an indication that the restriction site , genotyping can be carried out by performing subject is predisposed to Alzheimer ' s disease . The variant PCR using non - allele specific primers spanning the poly - alleles associated with Alzheimer ' s disease for each of the morphic site and digesting the resultant PCR product using 45 SNPs in the combinations described above are as follows: the appropriate restriction enzyme ( also known as PCR - rs798893 ( C ) , rs725106 ( A ), rs1341665 ( A ), rs1359059 ( A ) , RFLP ) . Restriction fragment length polymorphisms, includ - rs1532278 ( C ) , rs1801274 ( G ), rs2036108 ( T ) , rs811925 ing those resulting from the presence of a single nucleotide ( G ) , rs883524 ( C ) , rs 1065457 ( G ) , rs1148613 ( C ) , rs295 ( C ) , polymorphism , may be scored by digesting genomic DNA rs290258 (G ), rs365836 (G ) and rs569214 ( T ). with an appropriate enzyme then performing a Southern blot 50 Genotyping is preferably carried out in vitro , and is most using a labelled probe corresponding to the polymorphic preferably performed on an isolated sample containing region ( see Molecular Cloning : A Laboratory Manual, Sam - genomic DNA prepared from a suitable tissue sample brook , Fritsch and Maniatis , Cold Spring Harbor Labora obtained from the subject under test . Most commonly , tory , Cold Spring Harbor, N . Y .) . genomic DNA is prepared from a sample of whole blood or In the context of the present invention , " genotyping ” of 55 brain tissue, according to standard procedures which are any given polymorphic variant may advantageously com - well known in the art . If genomic sequence data for the prise screening for the presence or absence in the genome of individual under test in the region containing the SNP is the subject of both the normal or wild type allele and the available , for example in a genomic sequence database as a variant or mutant allele associated with disease , or may result of a prior genomic sequencing exercise , then geno comprise screening for the presence or absence of either 60 typing of the SNP may be accomplished by searching the individual allele , it generally being possible to draw con - available sequence data . clusions about the genotype of an individual at a polymor- In the case of genetic variants which have a detectable phic locus having two alternative allelic forms just by effect on the mRNA transcripts transcribed from a given screening for one or other of the specific alleles . gene , for example variants which cause altered splicing or Alzheimer' s disease is a complex and multi - factorial 65 which affect transcript termination or which affect the level condition . In any given individual the development of AD is or mRNA expression , then as an alternative to detecting the likely to be associated with accumulation of genetic varia presence of the variant at the genomic DNA level, the US 9 , 944 ,986 B2 22 presence of the variant may be inferred by evaluating the pathogenesis preceding formation of both amyloid - ß mRNA expression pattern using any suitable technique . plaques and neurofibrillary tangles . Therefore , detection of Similarly , in the case of genetic variants which have a these cell cycle changes may be used to assist with diagnosis detectable effect on the protein products encoded by a gene , of Alzheimer ' s disease at an early stage , even in asymptom for example variants which cause a change in primary amino 5 atic individuals . acid sequence, structure or properties of the encoded protein , Previous studies have shown that it is not cell cycle the presence of the variant may be inferred by evaluating the re - entry per se that contributes to Alzheimer ' s disease but sequence , structure or properties of the protein using any rather the inability of neurons from Alzheimer ' s disease convenient technique . patients to respond appropriately to this cell - cycle re - entry . The above - described screening methods may be used 10 In particular , neurons from Alzheimer ' s disease patients are prognostically to identify individuals pre - disposed to unable to initiate G1 arrest and subsequently undergo re Alzheimer' s disease ( AD ) by virtue of their genetic make differentiation , as a result of a defect in the G1/ S regulatory up . The “ pre- disposition to Alzheimer 's disease ” may be checkpoint. Furthermore , this regulatory defect at the G1/ S manifest as an increased risk of developing disease as transition occurs in cells other than neurons in individuals compared to the general population , or as an earlier age of 15 with Alzheimer ' s disease , for example lymphocytes . disease onset as compared to individuals who do not possess It is therefore possible to assist with the diagnosis of a variant allele associated with Alzheimer ' s disease . Alzheimer ' s disease in the live human subject by measuring In certain embodiments , the method may be used to the differential responsiveness of lymphocytes taken from screen asymptomatic individuals ( i . e . individuals who do the subject to G1 inhibitors, such as rapamycin . not exhibit significant symptoms of AD according to stan - 20 The present diagnostic methods are based on the geno dard diagnostic criteria ) in order to identify those “ at risk " typing of a subject to look for the presence of polymor of developing AD , and /or those likely to exhibit an earlier phisms, particularly single nucleotide polymorphisms age of onset of AD . The results of such screens may facilitate (SNPs ) in rapamycin - sensitive genes or in genes which early intervention with therapeutic treatments , particularly affect the expression of rapamycin - sensitive genes. Impor prophylactic treatments aimed at preventing , reducing or 25 tantly , it has been shown that SNPs within rapamycin delaying the clinical symptoms of Alzheimer ' s disease . sensitive genes or SNPs in genes which affect the expression In further embodiments the screening methods may be of rapamycin - sensitive genes correlate with the differential used to screen patients who exhibit clinical symptoms of response to rapamycin observed in lymphocytes collected Alzheimer ' s disease , for example to assist in correct diag - from individual Alzheimer ' s disease patients . Therefore , the nosis of AD and/ or to investigate the genetic basis of 30 present methods involve genotyping a subject for a poly suspected or confirmed AD . morphism in one or more of the rapamycin - sensitive genes Diagnostic Methods Based on Detection of Polymor - shown in Table 1 , or a polymorphism in one or more genes phisms which affect the expression of one or more of the rapamycin In a further aspect, the invention provides a method to sensitive genes shown in Table 1 as an independent means assist with diagnosis of Alzheimer ' s disease in a live human 35 to assist with diagnosis of Alzheimer ' s disease . In certain subject , which method comprises genotyping the subject for embodiments , the methods may involve genotyping a sub a polymorphism in one or more of the rapamycin - sensitive ject for multiple polymorphisms or polymorphic variants in genes shown in Table 1 , or a polymorphism in one or more one or more of the genes shown in Table 1 or multiple genes which affect the expression of one or more of the polymorphisms or polymorphic variants in one or more rapamycin - sensitive genes shown in Table 1 , wherein the 40 genes which affect the expression of one or more of the presence of at least one variant allele associated with rapamycin -sensitive genes shown in Table 1 , or a combina Alzheimer' s disease is indicative of Alzheimer 's disease. tion of both . In a preferred embodiment, genotyping of Embodiments of the invention described in the context of multiple polymorphisms in a single patient sample may be diagnostic methods of the invention based on measuring the carried out simultaneously , for example with the use of a expression level of one or more rapamycin - sensitive genes 45 microarray or “ gene chip ” . are equally applicable to this further aspect of the invention . In preferred embodiments, the method comprises geno In addition , embodiments of the invention described in the typing a subject for one or more polymorphisms in one or context ofmethods for assessing pre - disposition to Alzheim - more rapamycin - sensitive genes selected from LILRB2, er' s disease based on genotyping a subject for one or more FAM5C , CRP , CLU , FCGR2A , CD1E , FAM5C , LPL , SYK polymorphisms are equally applicable to this further aspect 50 and CUX1. Alternatively , or in addition , the method may of the invention . comprise genotyping a subject for one or more polymor Methods to assist with diagnosis of Alzheimer ' s disease in phisms in one or more genes which affect the expression of a live human subject have been described in International one or more rapamycin - sensitive genes , wherein the “ regu patent application no . WO02 /073212 , incorporated herein latory ” genes are selected from POU2F1 , ADRA1A , by reference . These methods comprise a step of screening 55 PRDM1 and LOXL2. non -neuronal cells from a human subject for the presence of In preferred embodiments , the diagnostic methods a cell cycle regulatory defect at the G1/ S transition . One of involve genotyping a subject for one or more , two or more , the ways in which this defect can be assessed is by mea - three or more , four or more , five or more , six or more , seven suring the responsiveness of the non - neuronal cells to a G1 or more , eight or more , nine or more , ten or more polymor inhibitor, for example rapamycin . Differential responsive - 60 phisms selected from the group of single nucleotide poly ness to a G1 inhibitor in lymphocytes taken from a subject morphisms consisting of rs798893 , rs725106 , rs 1341665 , suspected of having Alzheimer' s disease is indicative of rs1359059 , rs1532278 , rs1801274 , rs2036108 , rs811925 , disease . rs883524 , rs1065457 , rs1148613 , rs295 , rs290258 , The diagnostic test described in Wo02 / 073212 was rs365836 and rs569214 , as characterised in Table 14 . The developed based on the discovery that Alzheimer ' s disease 65 presence of at least one variant allele associated with is associated with aberrant re - entry of neurons into the cell Alzheimer ' s disease is an indication that the subject is division cycle . This change is an early event in disease pre - disposed to Alzheimer' s disease . The variant alleles US 9 ,944 ,986 B2 23 24 associated with Alzheimer ' s disease for the SNPs described The sample taken from the subject may be any sample above are as follows: rs798893 ( C ), rs725106 ( A ) , suitable for genetic analysis including but not limited to rs1341665 ( A ) , rs1359059 ( A ), rs1532278 ( C ) , rs1801274 blood , saliva, tears , urine, skin , hair or any other tissue ( G ) , rs2036108 ( T ) , rs811925 ( G ) , rs883524 ( C ), rs1065457 containing nucleic acid . (G ) , rs1148613 ( C ) , rs295 ( C ) , rs290258 ( G ) , rs365836 ( G ) 5 The array or kit may take any suitable format for the and rs569214 ( T ) . detection of polymorphisms, and the reagents forming the array or included in the kit will be dependent on the format The screening methods may also involve genotyping a adopted by the user . In certain embodiments , the array may subject for one or more, two or more, three or more, four or take the form of a microarray or " gene chip " wherein more , five or more , six or more , seven or more , eight or 10 oligonucleotides capable of detecting the one or more poly more , nine or more , ten or more polymorphisms selected morphisms of interest , if present within the sample , are from the group of polymorphisms consisting of polymor immobilised on a solid substrate . In certain embodiments , phisms, particularly SNPs, associated with or in linkage the oligonucleotides are allele - specific oligonucleotides disequilibrium (LD ) with the single nucleotide polymor capable of detecting the one or more polymorphisms by phisms: rs798893 , rs725106 , rs1341665, rs1359059$ 1339059 , 15 hybridisation to the variant allele . The invention therefore rs1532278 , rs1801274, rs2036108 , rs811925 , rs883524 , provides an array comprising multiple allele - specific oligo rs1065457 , rs1148613 , rs295 , rs290258 , rs365836 and nucleotides capable of detecting at least two, at least three , rs569214 . Polymorphisms “ associated ” with the SNPs in at least four, at least five etc different polymorphisms as Table 14 include polymorphisms in close proximity to the described elsewhere herein . The design of suitable allele characterised SNPs. Polymorphisms in linkage disequilib - 20 specific oligonucleotides or probes and the construction of rium with the characterised SNPs could be identified by one arrays comprising allele -specific oligonucleotides for detect of skill in the art using standard association mapping tech ing one or more polymorphisms, particularly SNPs, in a niques described in the art . sample could be carried out using standard techniques well In further preferred embodiments , the diagnostic methods known in the art. will involve genotyping a subject for a combination of single 25 Kits according to the present invention may include polynucleotide polymorphisms selected from the following: reagents suitable for carrying out allele -specific Q -PCR , in order to detect one or more polymorphisms. Allele -specific ( i ) rs1065457 , rs798893 and rs2036108 ; Q - PCR is a variation of the standard polymerase chain ( ii ) rs1065457 , rs1148613 , rs290258 , rs725106 and reaction , which can be used to identify SNPs in a sample rs 1341665 ; r5799893 30 containing nucleic acid . The reagents would include all ( iii ) rs1065457 , rs295 , rs1359059, rs1532278 , rs798893 , 30 standardcontaining PCR nu reagents (DNA polymerase , Tris -HC1 , ( NH4) 2 rs365836 , rs725106 and rs1341665 ; SO4, MgCl2 , Tween20 , NATP, dCTP, DTTP , NGTP ) and ( iv ) rs1148613, rs290258 , rs725106 , rs1341665 and suitable primers with 3 ' ends encompassing the SNP. Kits rs2036108 ; may also include allele -specific restriction enzymes, which ( v ) rs1065457 , rs1148613 , rs290258 , rs725106100 , 35za can be used to detect the presence of SNPs based on the rs 1341665 and rs2036108 ; digestion pattern produced when the restriction enzyme (vi ) rs1065457 , rs1359059 , rs725106 and rs2036108 ; or digests the nucleic acid sample , as described elsewhere ( vii ) rs798893 and rs725106 , herein . wherein the presence of a combination of variant alleles Methods to Monitor mTOR Signalling associated with Alzheimer ' s disease is indicative of a posi- 40 As discussed elsewhere herein , the polymorphisms to be tive Alzheimer ' s disease diagnosis . detected in the context of the screening and diagnostic As noted above, the diagnostic methods described herein methods described above , are either in one or more of the may also be used in combination or together with other rapamycin -sensitive genes shown in Table 1 , or in one or methods or tests used for Alzheimer ' s disease diagnosis , for more genes which affect the expression of one or more of the example in order to improve the specificity and / or sensitivity 45 rapamycin -sensitive genes shown in Table 1 . The inhibitor of these methods or tests . In a preferred embodiment, the rapamycin is known to inhibit the serine/ threonine kinase diagnostic method of the present invention involves a first mTOR in human cells and thereby reduce signalling down sterstep of genotyping aa subjectsubiect for a combination of polymor- stream of this protein . It follows therefore that genes iden phisms consisting of prl and pr10 and a second step of tified as rapamycin -sensitive genes are linked to mTOR b . 50 signalling in cells . determining plasma homocysteine levels in the same sub - 50 Therefore , in a further aspect, the present invention also ject. provides a method by which to monitormTOR signalling in Arrays and Kits a human cell, which method comprises detecting one or The present invention also provides arrays and kits for more polymorphisms in one or more of the rapamycin detecting one or more, two or more , three or more , four or sensitive genes shown in Table 1 , or one or more polymor more , five or more , six or more , seven or more, eight or phisms in one or more genes which affect the expression of more , nine or more , ten or more genetic polymorphisms or one or more of the rapamycin -sensitive genes shown in polymorphic variants in a sample taken from a subject . The Table 1 , wherein the presence of at least one variant allele one or more polymorphisms to be detected by the arrays and associated with a differential response to rapamycin is kits provided herein are in one or more of the rapamycin - 60 indicative of the status of mTOR signalling in the cell . sensitive genes shown in Table 1 , or in one or more genes The cytoplasmic kinase mTOR is stimulated or activated which affect the expression of one or more of the rapamycin - by a wide variety of upstream signals. These include signals sensitive genes shown in Table 1 . generated or triggered as a result of nutrient sensing, Embodiments of the invention already described above in hypoxia , and /or the activity of growth factors and their relation to the screening methods and diagnostic methods of 65 cognate receptors . Activation of mTOR upregulates its the invention relating to the use of polymorphisms are kinase activity and thereby increases mTOR -mediated phos equally applicable to the arrays and kits provided . phorylation of downstream protein targets within the cell. In US 9 , 944 ,986 B2 25 26 most cases, the direct downstream protein targets of mTOR taken to mean the functional integrity of signalling through interact with a variety of further molecular targets , and in the mTOR signalling pathway . Therefore , the presence of at doing so , stimulate a wide variety of cellular responses , such least one variant allele associated with a differential as increased protein synthesis and the promotion of cell response to rapamycin may be used to determine the status growth and proliferation . The chain of molecular events 5 of mTOR signalling in a cell . In certain embodiments , the triggered downstream of mTOR -mediated phosphorylation presence of at least one variant allele associated with a of its direct protein targets is defined herein as the “ mTOR differential response to rapamycin may be used to determine signalling pathway ” . that the functional integrity of the mTOR signalling pathway The methods of the present aspect of the invention allow is compromised in a particular cell . for the monitoring ofmTOR signalling. By “ monitoring ” is 10 Wherein the one or more polymorphisms is / are selected meant determination of the level of activity downstream of from the group consisting of rs798893 , rs725106 , the mTOR kinase , for example the level of activity of rs 1341665 , rs1359059 , rs1532278 , rs1801274 , rs2036108 , proteins present within the downstream signalling pathways . rs811925 , rs883524 , rs 1065457 , rs1148613 , rs295 , Monitoring may be carried out in particular , to determine the rs290258 , rs365836 and rs569214 , the bases of the variant functional integrity of the mTOR signalling pathway within 15 alleles associated with a differential response to rapamycin a human cell . are as follows: rs798893 ( C ) , rs725106 ( A ) , rs1341665 ( A ) , In preferred embodiments , the one or more polymor - rs1359059 ( A ), rs1532278 ( C ) , rs1801274 (G ), rs2036108 phisms for detection are in one or more of the rapamycin - ( T ) , rs811925 ( G ), rs883524 ( C ) , rs 1065457 ( G ), rs1148613 sensitive genes selected from LILRB2 , FAM5C , CRP, CLU , ( C ) , rs295 ( C ) , rs290258 ( G ) , rs365836 ( G ) and rs569214 FCGR2A , CD1E , FAM5C , LPL , SYK and CUX1 and / or in 20 ( T ) . one or more of the genes which affect the expression of one The methods of the present invention may be used to or more rapamycin - sensitive genes , selected from POU2F1, monitor mTOR signalling in any type of human cell. For ADRA1A , PRDM1 and LOXL2 . example , the human cell may be a cell pre - treated with a In preferred embodiments , the methods involve detecting compound such as a pharmacological inhibitor . In a pre one or more , two or more , three or more , four or more , five 25 ferred embodiment of the invention , the human cell is a or more , six or more , seven or more , eight or more , nine or lymphocyte . more , ten or more polymorphisms selected from the group Furthermore, in preferred embodiments of the invention , of single nucleotide polymorphisms consisting of rs798893 , the human cell may be taken from an individual or human rs725106 , rs 1341665 , rs1359059 , rs1532278 , rs1801274 , subject suspected of having a particular condition or disease , rs2036108 , rs811925 , rs883524 , rs 1065457 , rs1148613 , 30 or considered to be at risk of developing a particular disease , rs295 , rs290258 , rs365836 and rs569214 , as characterised in most preferably Alzheimer' s disease . Wherein the human Table 14 . The methods may also involve detecting one or subject is suspected of having or developing Alzheimer ' s more , two ormore , three or more , four or more, five or more , disease , the cell may be taken from a subject that is six or more , seven or more , eight or more , nine or more , ten asymptomatic for Alzheimer' s disease , or a subject who or more polymorphisms selected from the group of poly - 35 exhibits mild cognitive impairment or a subject exhibiting morphisms consisting of polymorphisms, particularly SNPs, one or more symptoms consistent with Alzheimer ' s disease . associated with or in linkage disequilibrium (LD ) with the The purpose of monitoring mTOR signalling in a cell single nucleotide polymorphisms: rs798893 , rs725106 , taken from an individual or subject suspected of having a rs1341665 , rs1359059 , rs1532278 , rs1801274 , rs2036108 , particular disease or considered at risk of a particular dis rs811925 , rs883524 , rs1065457 , rs1148613 , rs295 , 40 ease, may be to assist with diagnosis of disease in the subject rs290258 , rs365836 and rs569214 . or to assess the subject ' s pre - disposition to disease . In the As described elsewhere herein , cells from subjects with preferred embodiment of the present invention wherein the Alzheimer' s disease typically exhibit a differential response human cell is isolated from a human subject suspected of to G1/ S inhibitors, including rapamycin , as a result of a having Alzheimer ' s disease , themethod may be carried out defect in the G1/ S cell cycle transition . This differential 45 in order to assist with the diagnosis of Alzheimer ' s disease response is reported in for example , WO02 /073212 , incor - or to assess a subject' s pre- disposition to Alzheimer ' s dis porated herein by reference . The SNPs or variant alleles ease . characterised herein are associated with a differential The present methods may also be used to assist with response to rapamycin as seen in lymphocytes collected diagnosis of other diseases or conditions wherein dysregu from individuals with Alzheimer ' s disease . As noted above , 50 lation of signalling through the mTOR pathway is an under rapamycin responsiveness is indicative of the status of lying cause or consequence , for example cancer , type II mTOR signalling in the cell wherein " status" should be diabetes, dementia following brain injury or stroke . TABLE 1 Rapamycin -sen sensitive genes ( 1051 genes) Symbol Entrez Gene Name Location Type ( s ) A2M alpha - 2 -macroglobulin Extracellular Space transporter AADACL2 arylacetamide deacetylase - like 2 unknown other AASDH aminoadipate -semialdehyde dehydrogenase unknown enzyme ABCA8 ATP -binding cassette , sub - family A (ABC1 ) , member 8 Plasma Membrane transporter ABCB5 ATP -binding cassette , sub - family B (MDR / TAP ), Plasma Membrane transporter member 5 ABCD2 ATP -binding cassette , sub - family D ( ALD ) , member 2 Cytoplasm transporter ABHD2 abhydrolase domain containing 2 unknown enzyme ABI??? ABI family , member 3 (NESH ) binding protein Extracellular Space other ABLIM2 actin binding LIM protein family , member 2 Cytoplasm other US 9 ,944 ,986 B2 27 TABLE 1 - continued Rapamycin -sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type( s ) ABRA actin -binding Rho activating protein Cytoplasm transcription regulator ABTB1 ankyrin repeat and BTB (POZ ) domain containing 1 Cytoplasm translation regulator ACOX2 acyl- CoA oxidase 2 , branched chain Cytoplasm enzyme ???? acid phosphatase , prostate Extracellular Space phosphatase ACSL6 acyl- CoA synthetase long- chain family member 6 Cytoplasm enzyme ACSS1 acyl- CoA synthetase short- chain family member 1 Cytoplasm enzyme ACTRT1 actin - related protein T1 Cytoplasm other ACVR2B activin A receptor , type IIB Plasma Membrane kinase ADAMTS13 ADAM metallopeptidase with thrombospondin type 1 Extracellular Space peptidase motif, 13 ADAMTS15 ADAM metallopeptidase with thrombospondin type 1 Extracellular Space peptidase motif, 15 ADAMTS16 ADAM metallopeptidase with thrombospondin type 1 Extracellular Space other motif , 16 ADAMTS2 ADAM metallopeptidase with thrombospondin type 1 Extracellular Space peptidase motif, 2 ADAMTS20 ADAM metallopeptidase with thrombospondin type 1 Extracellular Space peptidase motif , 20 ADAMTS3 ADAM metallopeptidase with thrombospondin type 1 Extracellular Space peptidase motif , 3 ADAMTS9 ADAM metallopeptidase with thrombospondin type 1 Extracellular Space peptidase motif , 9 ADAMTSL3 ADAMTS - like 3 unknown other ADAMTSL4 ADAMTS - like 4 Extracellular Space other ADAMTSL5 ADAMTS - like 5 Extracellular Space other ADH6 ( includes EG : 130 ) alcohol dehydrogenase 6 ( class V ) Cytoplasm enzyme ADIPOQ adiponectin , C1Q and collagen domain containing Extracellular Space other ADSSL1 adenylosuccinate synthase like 1 Cytoplasm enzyme AGT angiotensinogen ( serpin peptidase inhibitor, clade A , Extracellular Space growth factor member 8 ) AGTR1 angiotensin II receptor , type 1 Plasma Membrane G -protein coupled receptor AHNAK AHNAK nucleoprotein Nucleus other AHNAK2 AHNAK nucleoprotein 2 unknown other AKR1D1 aldo -keto reductase family 1 , member D1 ( delta 4 - 3 Cytoplasm enzyme ketosteroid - 5 -beta - reductase ) ALDH1A3 aldehyde dehydrogenase 1 family , member A3 Cytoplasm enzyme AMY1A ( includes others ) amylase , alpha 1A ( salivary ) Extracellular Space enzyme ANGPT2 angiopoietin 2 Extracellular Space growth factor ANGPTL2 angiopoietin - like 2 Extracellular Space other ANGPTL5 angiopoietin -like 5 Extracellular Space other ANK1 ankyrin 1 , erythrocytic Plasma Membrane other ANKRD12 ankyrin repeat domain 12 Nucleus other ANKRD36B (includes ankyrin repeat domain 36B Nucleus transcription others ) regulator ANKRD36B ( includes ankyrin repeat domain 36B Nucleus transcription others ) regulator ANKRD42 ankyrin repeat domain 42 Nucleus transcription regulator ANKRD45 ankyrin repeat domain 45 Nucleus transcription regulator ANKRD50 ankyrin repeat domain 50 unknown other ANKRD6 ankyrin repeat domain 6 Nucleus transcription regulator ANKSIB ankyrin repeat and sterile alpha motif domain Nucleus other containing 1B ANO3 anoctamin 3 unknown other ANXA10 annexin A10 Cytoplasm other ANXA2R annexin A2 receptor Plasma Membrane other AP1S1 adaptor - related protein complex 1 , sigma 1 subunit Cytoplasm transporter APBB1IP amyloid beta ( A4 ) precursor protein - binding , family B , Cytoplasm other member 1 interacting protein ???? apolipoprotein B (including Ag( x ) antigen ) Extracellular Space transporter APOBEC3G apolipoprotein B mRNA editing enzyme, catalytic Nucleus enzyme polypeptide - like 3G APOLD1 apolipoprotein L domain containing 1 unknown other AQP12A /AQP12B aquaporin 12B Cytoplasm transporter AQP4 aquaporin 4 Plasma Membrane transporter AQP9 aquaporin 9 Plasma Membrane transporter AR androgen receptor Nucleus ligand dependent nuclear receptor US 9 ,944 ,986 B2 30 TABLE 1 - continued Rapamycin- sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type( s ) ARHGAP28 Rho GTPase activating protein 28 Cytoplasm other ARHGEF4 Rho guanine nucleotide exchange factor (GEF ) 4 Cytoplasm other ARHGEF6 Rac/ Cdc42 guanine nucleotide exchange factor Cytoplasm other (GEF ) 6 ARL14 ADP - ribosylation factor - like 14 unknown other ARL17B /LOC100294341 ADP- ribosylation factor - like 17B unknown other ARMC2 armadillo repeat containing 2 unknown other ARSE arylsulfatase E (chondrodysplasia punctata 1 ) Cytoplasm enzyme AS3MT arsenic ( + 3 oxidation state ) methyltransferase Cytoplasm enzyme ASB11 ankyrin repeat and SOCS box containing 11 Nucleus transcription regulator ASPM asp ( abnormal spindle ) homolog, microcephaly Nucleus other associated ( Drosophila ) ASXL3 additional sex combs like 3 ( Drosophila ) unknown other ATAD3A ATAD3B ATPase family , AAA domain containing 3A Nucleus other ATFTIP activating transcription factor 7 interacting protein Nucleus transcription regulator ATP13A4 ATPase type 13A4 unknown transporter ATP6VOD2 ATPase , H + transporting, lysosomal 38 kDa, VO Cytoplasm transporter subunit d2 ATP8B3 ATPase , aminophospholipid transporter, class I , type Cytoplasm transporter 8B , member 3 ATRNL1 attractin - like 1 unknown other AUTS2 autism susceptibility candidate 2 unknown other B2M beta - 2 -microglobulin Plasma Membrane transmembrane receptor B3GALT4 UDP -Gal :betaGlcNAc beta 1, 3 - galactosyltransferase , Cytoplasm enzyme polypeptide 4 B4GALT6 UDP- Gal : betaGlcNAc beta 1 , 4 Cytoplasm enzyme galactosyltransferase , polypeptide 6 BAIAP2 BAIl -associated protein 2 Plasma Membrane kinase BAIAP2L2 BAIl -associated protein 2 - like 2 Cytoplasm other BARX2 BARX homeobox 2 Nucleus transcription regulator ????? BRCA2 and CDKN1A interacting protein Nucleus untukbutIaltiallewillhasamanother BCL2 B - cell CLL /lymphoma 2 Cytoplasm transporter BEGAIN brain - enriched guanylate kinase - associated homolog Nucleus other ( rat) BEND2 BEN domain containing 2 unknown other BEND6 BEN domain containing 6 unknown other BEST3 bestrophin 3 Nucleus ion channel BMP7 bone morphogenetic protein 7 Extracellular Space growth factor BMX BMX non -receptor tyrosine kinase Cytoplasm kinase BNC1 basonuclin 1 Nucleus transcription regulator BPESC1 blepharophimosis , epicanthus inversus and ptosis , unknown other candidate 1 (non - protein coding) BPI bactericidal/ permeability - increasing protein Plasma Membrane transporter BPIFB1 BPI fold containing family B , member 1 Extracellular Space other BRIP BRCA1 interacting protein C - terminal helicase 1 Nucleus enzyme BSN bassoon (presynaptic cytomatrix protein ) Plasma Membrane other BSND Bartter syndrome, infantile , with sensorineural Plasma Membrane ion channel deafness ( Barttin ) BSPRY B -box and SPRY domain containing Cytoplasm other BTNL9 butyrophilin - like 9 unknown other BVES blood vessel epicardial substance Plasma Membrane other C10orf10 chromosome 10 open reading frame 10 Cytoplasm other C10orf107 chromosome 10 open reading frame 107 unknown other C10orf111 chromosome 10 open reading frame 111 unknown other C11 orf67 chromosome 11 open reading frame 67 unknown other Cilorf87 chromosome 11 open reading frame 87 unknown other Cilorf88 chromosome 11 open reading frame 88 unknown other C12orf42 chromosome 12 open reading frame 42 unknown other C15orf43 chromosome 15 open reading frame 43 unknown other C15orf48 chromosome 15 open reading frame 48 Nucleus other C17orf78 chromosome 17 open reading frame 78 unknown other C17orf99 chromosome 17 open reading frame 99 unknown other C18orf26 chromosome 18 open reading frame 26 unknown other Clorf110 chromosome 1 open reading frame 110 unknown other Clorf127 chromosome 1 open reading frame 127 unknown other Clorf173 chromosome 1 open reading frame 173 unknown other Clorf226 chromosome 1 open reading frame 226 unknown other Clorf87 chromosome 1 open reading frame 87 unknown other C1QTNF6 Clq and tumor necrosis factor related protein 6 Extracellular Space other C20orf132 chromosome 20 open reading frame 132 unknown other C20orf85 chromosome 20 open reading frame 85 unknown other US 9 ,944 ,986 B2 31 TABLE 1 - continued Rapamycin - sensitive genes ( 1051 genes) Symbol Entrez Gene Name Location Type ( s ) C2orf16 chromosome 2 open reading frame 16 unknown other C3orf36 chromosome 3 open reading frame 36 unknown other C3orf70 chromosome 3 open reading frame 70 unknown other C4orf19 chromosome 4 open reading frame 19 unknown other C4orf22 chromosome 4 open reading frame 22 unknown other C4orf26 chromosome 4 open reading frame 26 unknown other C4orf36 chromosome 4 open reading frame 36 unknown other Coorf223 chromosome 6 open reading frame 223 unknown other C7orf41 chromosome 7 open reading frame 41 unknown other Clorf42 chromosome 8 open reading frame 42 unknown other CA12 carbonic anhydrase XII Plasma Membrane enzyme CA6 carbonic anhydrase VI Extracellular Space enzyme CACNA1D calcium channel , voltage -dependent , L type, alpha Plasma Membrane ion channel 1D subunit CADPS Ca + + -dependent secretion activator Plasma Membrane other CALD1 caldesmon 1 Cytoplasm other CARD14 caspase recruitment domain family , member 14 Cytoplasm other CATSPER2 cation channel , sperm associated 2 Plasma Membrane ion channel CATSPERD catsper channel auxiliary subunit delta unknown other CAV2 caveolin 2 Plasma Membrane other CBX7 chromobox homolog 7 Nucleus other CC2D2A coiled -coil and C2 domain containing 2A unknown other CCDC141 coiled - coil domain containing 141 unknown other CCDC28A coiled - coil domain containing 28A unknown other CCDC34 coiled - coil domain containing 34 unknown other CCDC40 coiled - coil domain containing 40 unknown other CCDC85A coiled - coil domain containing 85A unknown other CCL1 chemokine ( C - C motif ) ligand 1 Extracellular Space cytokine CCL11 chemokine ( C - C motif ) ligand 11 Extracellular Space cytokine CCL2 chemokine ( C - C motif ) ligand 2 Extracellular Space cytokine CCL26 chemokine ( C - C motif ) ligand 26 Extracellular Space cytokine CCLS chemokine ( C - C motif ) ligand 8 Extracellular Space cytokine CCNB2 cyclin B2 Cytoplasm other CCNG2 cyclin G2 Nucleus other CCR1 chemokine ( C - C motif ) receptor 1 Plasma Membrane G -protein coupled receptor CCR2 chemokine ( C - C motif ) receptor 2 Plasma Membrane G - protein coupled receptor CD177 CD177 molecule Cytoplasm other CDIA CDla molecule Plasma Membrane other CD1B CDlb molecule Plasma Membrane other CDIE CDle molecule Cytoplasm other CD44 ( includes CD44 molecule ( Indian blood group ) Plasma Membrane enzyme EG : 100330801 ) CD69 CD69 molecule Plasma Membrane transmembrane receptor CD96 CD96 molecule Plasma Membrane other CDC14B CDC14 cell division cycle 14 homolog B ( S . cerevisiae ) Nucleus phosphatase CDCP1 CUB domain containing protein 1 Plasma Membrane other CDCP2 CUB domain containing protein 2 unknown transporter CDH1 cadherin 1 , type 1 , E - cadherin ( epithelial) Plasma Membrane other CDH13 cadherin 13, H - cadherin ( heart ) Plasma Membrane other CDH26 cadherin 26 Plasma Membrane other CDH7 cadherin 7 , type 2 Plasma Membrane other CDHR1 cadherin -related family member 1 Plasma Membrane other CDKN1B cyclin -dependent kinase inhibitor 1B (p27 , Kipl ) Nucleus kinase CDKNIC cyclin - dependent kinase inhibitor 1C (p57 , Kip2 ) Nucleus other CDKN2C cyclin - dependent kinase inhibitor 2C (p18 , inhibits Nucleus transcription CDK4) regulator CDKN2D cyclin -dependent kinase inhibitor 2D (p19 , inhibits Nucleus transcription CDK4 ) regulator CDON Cdon homolog (mouse ) Plasma Membrane other CEACAM1 (includes carcinoembryonic antigen -related cell adhesion Plasma Membrane transmembrane others ) molecule 1 (biliary glycoprotein ) receptor CELF3 CUGBP, Elav- like family member 3 Nucleus transcription regulator CELF4 CUGBP , Elav- like family member 4 Nucleus translation regulator CELF6 CUGBP, Elav- like family member 6 unknown other CEP68 centrosomal protein 68 kDa Cytoplasm other CFHR5 complement factor H -related 5 Extracellular Space other CFTR cystic fibrosis transmembrane conductance regulator Plasma Membrane ion channel (ATP -binding cassette sub - family C , member 7 ) US 9 ,944 ,986 B2 33 34 TABLE 1 - continued Rapamycin -sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type ( s ) CGA glycoprotein hormones , alpha polypeptide Extracellular Space other CGNL1 cingulin - like 1 Plasma Membrane other CHD2 chromodomain helicase DNA binding protein 2 Nucleus enzyme CHP2 calcineurin B homologous protein 2 Cytoplasm other CHRDL2 chordin - like 2 Extracellular Space other CHRNAS cholinergic receptor, nicotinic , alpha 9 (neuronal ) Plasma Membrane transmembrane receptor ??? creatine kinase , muscle Cytoplasm kinase CLC Charcot- Leyden crystal protein Cytoplasm enzyme CLCA2 chloride channel accessory 2 Plasma Membrane ion channel CLEC7A C -type lectin domain family 7 , member A Plasma Membrane transmembrane receptor CLTB clathrin , light chain B Plasma Membrane other CLVS1 clavesin 1 Cytoplasm other CNRIP1 cannabinoid receptor interacting protein 1 unknown other CNTN3 contactin 3 (plasmacytoma associated ) Plasma Membrane other COBL cordon - bleu homolog (mouse ) unknown other COL11A1 collagen , type XI, alpha 1 Extracellular Space other COL13A1 collagen , type XIII , alpha 1 Plasma Membrane other COL1A2 collagen , type I, alpha 2 Extracellular Space other COL6A3 collagen , type VI, alpha 3 Extracellular Space other COL6A6 collagen , type VI, alpha 6 Extracellular Space other COL8A2 collagen , type VIII , alpha 2 Extracellular Space other COMMD6 COMM domain containing 6 unknown other CORIN corin , serine peptidase Plasma Membrane peptidase CORO2A coronin , actin binding protein , 2A Cytoplasm other CPB2 carboxypeptidase B2 (plasma ) Extracellular Space peptidase CPE ( includes carboxypeptidase E Plasma Membrane peptidase EG : 12876 ) CPLX2 complexin 2 Cytoplasm other CPXM2 carboxypeptidase X (M14 family ) , member 2 Extracellular Space peptidase CRB1 crumbs homolog 1 ( Drosophila ) Plasma Membrane other CREB3L4 CAMP responsive element binding protein 3 - like 4 Nucleus transcription regulator CREBRF CREB3 regulatory factor unknown other CRIM1 cysteine rich transmembrane BMP regulator 1 Extracellular Space kinase ( chordin - like ) CROT carnitine O - octanoyltransferase Cytoplasm enzyme CRP C - reactive protein , pentraxin - related Extracellular Space other CRTAC1 cartilage acidic protein 1 Extracellular Space other CRYBG3 beta -gamma crystallin domain containing 3 unknown other CRYGB crystallin , gamma B Nucleus other CRYGD crystallin , gamma D Cytoplasm other CSDC2 cold shock domain containing C2 , RNA binding Cytoplasm other CSFIR colony stimulating factor 1 receptor Plasma Membrane kinase CSN2 casein beta Extracellular Space kinase CTAGIB ( includes cancer/ testis antigen 1B Cytoplasm other others ) CTDSP1 CTD ( carboxy - terminal domain , RNA polymerase II, Nucleus phosphatase polypeptide A ) small phosphatase 1 CTNNA3 catenin ( cadherin -associated protein ), alpha 3 Plasma Membrane other CTSF cathepsin F Cytoplasm peptidase CXorf51 A / CXorf51B chromosome X open reading frame 51A unknown other CYP2C9 cytochrome P450 , family 2 , subfamily C , polypeptide 9 Cytoplasm enzyme CYP3A5 cytochrome P450 , family 3 , subfamily A , polypeptide 5 Cytoplasm enzyme CYP4F11 cytochrome P450 , family 4 , subfamily F , polypeptide Cytoplasm enzyme 11 CYP4F2 cytochrome P450 , family 4 , subfamily F , polypeptide 2 Cytoplasm enzyme CYP4F3 cytochrome P450 , family 4 , subfamily F , polypeptide 3 Cytoplasm enzyme CYP4V2 cytochrome P450 , family 4 , subfamily V , polypeptide 2 Cytoplasm enzyme CYP4X1 cytochrome P450 , family 4 , subfamily X , polypeptide 1 Cytoplasm enzyme CYP4Z1 cytochrome P450 , family 4 , subfamily Z , polypeptide 1 Cytoplasm enzyme CYR61 cysteine - rich , angiogenic inducer, 61 Extracellular Space other CYSLTR2 cysteinyl leukotriene receptor 2 Plasma Membrane G -protein coupled receptor DAOA D -amino acid oxidase activator Cytoplasm other DAPL1 death associated protein - like 1 unknown other DCAF12L1 DDB1 and CUL4 associated factor 12- like 1 unknown other DCC deleted in colorectal carcinoma Plasma Membrane transmembrane ullamthullreceptor US 9 ,944 ,986 B2 35 36 TABLE 1 - continued Rapamycin - sensitive genes ( 1051 genes) Symbol Entrez Gene Name Location Type( s ) DCD dermcidin Extracellular Space other DCLK1 doublecortin - like kinase 1 Plasma Membrane kinase DCN decorin Extracellular Space other DDI1 ( includes DNA -damage inducible 1 homolog 1 ( S . cerevisiae ) unknown other EG : 367012 ) DDX17 DEAD ( Asp - Glu - Ala - Asp ) box helicase 17 Nucleus enzyme DEFA4 defensin , alpha 4 , corticostatin Extracellular Space other DEFB119 defensin , beta 119 Extracellular Space other DHRS12 dehydrogenase / reductase ( SDR family ) member 12 Nucleus other DHRS3 dehydrogenase/ reductase (SDR family) member 3 Cytoplasm enzyme DKFZP586K1520 DKFZP586K1520 protein unknown other DLEUT deleted in lymphocytic leukemia , 7 unknown other DLG2 discs, large homolog 2 (Drosophila ) Plasma Membrane kinase DLGAP4 discs , large (Drosophila ) homolog - associated protein 4 Plasma Membrane other DLX2 distal - less homeobox 2 Nucleus transcription regulator DMRTB1 DMRT- like family B with proline - rich C - terminal, 1 Nucleus transcription regulator DMRTC1/ DMRTC1B DMRT -like family C1B unknown other DMRTC2 DMRT- like family C2 Nucleus transcription regulator DNAH1 dynein , axonemal, heavy chain 1 unknown other DNAH6 dynein , axonemal, heavy chain 6 unknown other DNAJB7 DnaJ (Hsp40 ) homolog, subfamily B , member 7 unknown other DPY19L1P1 dpy - 19 - like 1 pseudogene 1 ( C . elegans) unknown other DRP2 dystrophin related protein 2 Plasma Membrane other DSEL dermatan sulfate epimerase - like unknown enzyme DST dystonin Plasma Membrane other DTD1 D - tyrosyl- tRNA deacylase 1 homolog ( S . cerevisiae ) Cytoplasm enzyme DUSP13 dual specificity phosphatase 13 Cytoplasm phosphatase DUSP21 dual specificity phosphatase 21 Cytoplasm phosphatase DUSP27 dual specificity phosphatase 27 (putative ) unknown phosphatase DYNCZLI1 dynein , cytoplasmic 2 , light intermediate chain 1 Cytoplasm other DYNLRB1 dynein , light chain , roadblock - type 1 Cytoplasm other DZIPIL DAZ interacting protein 1 - like unknown other DZIP3 DAZ interacting protein 3 , zinc finger Cytoplasm enzyme EBF2 early B - cell factor 2 Nucleus other EBF3 early B - cell factor 3 Nucleus other ECHDC2 enoyl CoA hydratase domain containing 2 unknown shilihubunother ECM2 ( includes extracellular matrix protein 2 , female organ and Extracellular Space other EG : 1842 ) adipocyte specific ECT2L epithelial cell transforming sequence 2 oncogene- like unknown other EDDM3A epididymal protein 3A Extracellular Space other EDDM3B epididymal protein 3B Extracellular Space other EEPD1 endonuclease / exonuclease /phosphatase family unknown other domain containing 1 EFCAB3 EF -hand calcium binding domain 3 unknown other EGF ( includes epidermal growth factor Extracellular Space growth factor EG : 13645 ) EGLN3 egl nine homolog 3 ( C . elegans) Cytoplasm enzyme EGR1 early growth response 1 Nucleus transcription regulator ELAVL3 ELAV ( embryonic lethal, abnormal vision , Nucleus other Drosophila ) - like 3 (Hu antigen C ) ELAVL4 ELAV ( embryonic lethal, abnormal vision , Cytoplasm other Drosophila ) - like 4 (Hu antigen D ) ELOVL7 ELOVL fatty acid elongase 7 Cytoplasm enzyme EMCN endomucin Extracellular Space other EMX2OS EMX2 opposite strand /antisense RNA (non - protein unknown other coding) ENPEP glutamyl aminopeptidase ( aminopeptidase A ) Plasma Membrane peptidase ENPP6 ectonucleotide pyrophosphatase /phosphodiesterase 6 Cytoplasm enzyme ENTPD8 ectonucleoside triphosphate diphosphohydrolase 8 unknown enzyme EPB41L4B erythrocyte membrane protein band 4 . 1 like 4B unknown transporter EPHA5 EPH receptor A5 Plasma Membrane kinase EPO erythropoietin Extracellular Space cytokine ERBB3 ( includes V - erb -b2 erythroblastic leukemia viral oncogene Plasma Membrane kinase EG : 13867) homolog 3 (avian ) ERBB4 V - erb - a erythroblastic leukemia viral oncogene Plasma Membrane kinase homolog 4 (avian ) ESR1 estrogen receptor 1 Nucleus ligand dependent nuclear receptor ESYT3 extended synaptotagmin - like protein 3 unknown other EYA1 eyes absent homolog 1 (Drosophila ) Nucleus phosphatase US 9 ,944 ,986 B2 37 38 TABLE 1 - continued Rapamycin -sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type( s ) F11 coagulation factor XI Extracellular Space peptidase F2 coagulation factor II ( thrombin ) Extracellular Space peptidase F2RL2 coagulation factor II ( thrombin ) receptor- like 2 Plasma Membrane G - protein coupled receptor F9 coagulation factor IX Extracellular Space peptidase FAM100B family with sequence similarity 100 , member B unknown other FAM135B family with sequence similarity 135 , member B unknown enzyme FAM13C family with sequence similarity 13 , member C unknown other FAM149B1 family with sequence similarity 149 , member B1 unknown other FAM153A / FAM153B family with sequence similarity 153 , member A unknown other FAM155A family with sequence similarity 155 , member A unknown other FAM162B family with sequence similarity 162 , member B unknown other FAM171B family with sequence similarity 171 , member B unknown other FAM172A family with sequence similarity 172 , member A Cytoplasm transcription regulator FAM177A1 family with sequence similarity 177 , member Al unknown other FAM181B family with sequence similarity 181 , member B unknown other FAM19A1 family with sequence similarity 19 ( chemokine ( C - C unknown other motif) - like ) , member Al FAM211A family with sequence similarity 211 , member A unknown other FAM24A family with sequence similarity 24 , member A unknown other FAM26D family with sequence similarity 26 , member D unknown other FAM27E3 ( includes family with sequence similarity 27 , member E3 unknown other others ) FAM43B family with sequence similarity 43, member B unknown other FAM5C family with sequence similarity 5 , member C Cytoplasm other FAM64A family with sequence similarity 64 , member A Nucleus other FAM71D family with sequence similarity 71 , member D unknown other FAM74A3 family with sequence similarity 74 , member A3 unknown other FAM84A family with sequence similarity 84 , member A unknown other FAM92B family with sequence similarity 92, member B unknown other FAT3 FAT tumor suppressor homolog 3 (Drosophila ) unknown other FAXC failed axon connections homolog (Drosophila ) unknown other FBLN1 fibulin 1 Extracellular Space other FBN2 ( includes fibrillin 2 Extracellular Space other EG : 14119 ) FBP2 fructose - 1 ,6 -bisphosphatase 2 Cytoplasm phosphatase FBXL16 F -box and leucine- rich repeat protein 16 unknown other FCGBP Fc fragment of IgG binding protein Extracellular Space other FGB ( includes fibrinogen beta chain Extracellular Space other EG : 110135 ) FGF11 fibroblast growth factor 11 Extracellular Space growth factor FGF18 fibroblast growth factor 18 Extracellular Space growth factor FGF2 fibroblast growth factor 2 (basic ) Extracellular Space growth factor FGF23 fibroblast growth factor 23 Extracellular Space growth factor FGF7 fibroblast growth factor 7 Extracellular Space growth factor FGFR1 fibroblast growth factor receptor 1 Plasma Membrane kinase FGG fibrinogen gamma chain Extracellular Space other FGGY FGGY carbohydrate kinase domain containing unknown other FGR Gardner- Rasheed feline sarcoma viral ( v - fgr ) Nucleus kinase oncogene homolog FIGN fidgetin Nucleus other FILIP1 filamin A interacting protein 1 Cytoplasm other FJX1 four jointed box 1 (Drosophila ) Extracellular Space other FLJ35946 uncharacterized protein FLJ35946 unknown other FLJ36000 uncharacterized FLJ36000 unknown other FLJ37035 uncharacterized LOC399821 unknown other FLJ37644 uncharacterized LOC400618 unknown other FLJ42875 uncharacterized LOC440556 unknown other FMN2 formin 2 unknown other FMO3 flavin containing monooxygenase 3 Cytoplasm enzyme FN1 fibronectin 1 Extracellular Space enzyme FN3K fructosamine 3 kinase Cytoplasm kinase FNBP1 formin binding protein 1 Nucleus enzyme FNDC5 fibronectin type III domain containing 5 unknown other FRAT2 frequently rearranged in advanced T -cell lymphomas 2 Cytoplasm other FREM3 FRAS1 related extracellular matrix 3 Extracellular Space other FRMD4A FERM domain containing 4A Plasma Membrane other FRMD6 FERM domain containing 6 Cytoplasm other FRMD7 FERM domain containing 7 Plasma Membrane other FSTL5 follistatin - like 5 Extracellular Space other FUTO fucosyltransferase 9 (alpha ( 1 , 3 ) fucosyltransferase ) Cytoplasm enzyme GOS2 GO /G1switch 2 Cytoplasm other US 9 ,944 ,986 B2 39 TABLE 1 - continued Rapamycin- sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type( s ) GAB1 GRB2 - associated binding protein 1 Cytoplasm other GABBR2 gamma- aminobutyric acid (GABA ) B receptor, 2 Plasma Membrane G -protein coupled receptor GABRA1 gamma - aminobutyric acid (GABA ) A receptor, alpha 1 Plasma Membrane ion channel GABRAS gamma- aminobutyric acid (GABA ) A receptor, alpha 5 Plasma Membrane ion channel GABRG1 gamma- aminobutyric acid (GABA ) A receptor , Plasma Membrane ion channel gamma 1 GABRR2 gamma - aminobutyric acid (GABA ) A receptor, rho 2 Plasma Membrane ion channel GAGE1 ( includes G antigen 1 unknown other others ) GALNT10 UDP - N - acetyl- alpha - D - galactosamine :polypeptide N Cytoplasm enzyme acetylgalactosaminyltransferase 10 (GalNAc - T10 ) GALNTS UDP - N - acetyl- alpha - D - galactosamine :polypeptide N Cytoplasm enzyme acetylgalactosaminyltransferase 5 (GalNAC - T5 ) GBP3 guanylate binding protein 3 Cytoplasm enzyme GCM2 glial cells missing homolog 2 (Drosophila ) Nucleus transcription regulator GDA guanine deaminase Cytoplasm enzyme GFAP glial fibrillary acidic protein Cytoplasm other GFRA1 GDNF family receptor alpha 1 Plasma Membrane transmembrane receptor GFRA2 GDNF family receptor alpha 2 Plasma Membrane transmembrane receptor GHRLOS ghrelin opposite strand /antisense RNA ( non - protein unknown other coding) GIGYF1 GRB10 interacting GYF protein 1 unknown other GIPR gastric inhibitory polypeptide receptor Plasma Membrane G -protein coupled receptor GLI2 GLI family zinc finger 2 Nucleus transcription regulator GLPIR glucagon - like peptide 1 receptor Plasma Membrane G -protein coupled receptor GLRA1 glycine receptor , alpha 1 Plasma Membrane ion channel GNAL guanine nucleotide binding protein ( G protein ), alpha Cytoplasm enzyme activating activity polypeptide , olfactory type GNG8 guanine nucleotide binding protein ( G protein ), Plasma Membrane gamma 8 GPAM glycerol- 3 - phosphate acyltransferase , mitochondrial Cytoplasm enzyme GPC3 glypican 3 Plasma Membrane other GPR110 G protein - coupled receptor 110 Plasma Membrane G - protein coupled receptor GPR116 G protein - coupled receptor 116 Plasma Membrane G -protein coupled receptor GPR176 G protein -coupled receptor 176 Plasma Membrane G -protein coupled receptor GPR34 G protein -coupled receptor 34 Plasma Membrane G -protein coupled receptor GPR37 G protein -coupled receptor 37 ( endothelin receptor Plasma Membrane G - protein type B - like ) coupled receptor GPR4 G protein - coupled receptor 4 Plasma Membrane G -protein coupled receptor GPX6 glutathione peroxidase 6 (olfactory ) Extracellular Space enzyme GPX8 glutathione peroxidase 8 (putative ) unknown enzyme GRIA1 glutamate receptor, ionotropic , AMPA 1 Plasma Membrane ion channel GRIA2 glutamate receptor, ionotropic , AMPA 2 Plasma Membrane ion channel GRIA3 glutamate receptor , ionotropic , AMPA 3 Plasma Membrane ion channel GRIK2 glutamate receptor , ionotropic , kainate 2 Plasma Membrane ion channel GRIK3 glutamate receptor, ionotropic , kainate 3 Plasma Membrane ion channel GRM1 glutamate receptor, metabotropic 1 Plasma Membrane G - protein coupled receptor GRPR gastrin -releasing peptide receptor Plasma Membrane G -protein coupled receptor GSGIL GSG1 - like unknown other GSN gelsolin Extracellular Space other GSTAD glutathione S - transferase alpha 1 Cytoplasm enzyme US 9 ,944 ,986 B2 41 TABLE 1 - continued Rapamycin- sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type (s ) GSTA5 glutathione S - transferase alpha 5 Cytoplasm enzyme GSTM3 glutathione S - transferase mu 3 (brain ) Cytoplasm enzyme GUCY1A3 guanylate cyclase 1 , soluble , alpha 3 Cytoplasm enzyme GUCY2F guanylate cyclase 2F , retinal Plasma Membrane kinase GVINP1 GTPase , very large interferon inducible pseudogene 1 Cytoplasm other HABP2 hyaluronan binding protein 2 Extracellular Space peptidase HAND1 heart and neural crest derivatives expressed 1 Nucleus transcription regulator HAND2 heart and neural crest derivatives expressed 2 Nucleus transcription regulator HAPLN1 hyaluronan and proteoglycan link protein 1 Extracellular Space other HCAR3 hydroxycarboxylic acid receptor 3 Plasma Membrane G -protein coupled receptor ??? hemopoietic cell kinase Cytoplasm kinase HDAC5 histone deacetylase 5 Nucleus transcription regulator HDACE histone deacetylase 9 Nucleus transcription regulator HESX1 HESX homeobox 1 Nucleus transcription regulator HGF hepatocyte growth factor ( hepapoietin A ; scatter Extracellular Space growth factor factor) HHIP hedgehog interacting protein Plasma Membrane other HHIPL1 HHIP - like 1 unknown other HIST1H2BN histone cluster 1 , H2bn Nucleus other HIST1H4A ( includes histone cluster 1 , H4a Nucleus other mothers ) HIVEP2 human immunodeficiency virus type I enhancer Nucleus transcription binding protein 2 regulator HMGCS2 3 -hydroxy - 3 -methylglutaryl - CoA synthase 2 Cytoplasm enzyme (mitochondrial ) HNF4G hepatocyte nuclear factor 4 , gamma Nucleus transcription regulator HOXA2 homeobox A2 Nucleus transcription regulator HOXAS homeobox A9 Nucleus transcription regulator HOXC8 homeobox C8 Nucleus transcription regulator HOXD10 homeobox D10 Nucleus transcription regulator HRASLS5 HRAS- like suppressor family , member 5 unknown other HSD17B13 hydroxysteroid (17 -beta ) dehydrogenase 13 Extracellular Space enzyme HTN3 histatin 3 Extracellular Space other HTR2C 5 -hydroxytryptamine ( serotonin ) receptor 2C , G Plasma Membrane G -protein protein - coupled coupled receptor HTR3C 5 -hydroxytryptamine ( serotonin ) receptor 3C , Plasma Membrane ion channel ionotropic HYDIN HYDIN , axonemal central pair apparatus protein unknown other IFFO1 intermediate filament family orphan 1 unknown other IFIT2 interferon- induced protein with tetratricopeptide Cytoplasm other repeats 2 IFNA16 interferon , alpha 16 Extracellular Space cytokine IGBP1 immunoglobulin ( CD79A ) binding protein 1 Cytoplasm phosphatase IGF2 insulin - like growth factor 2 (somatomedin A ) Extracellular Space growth factor IKZF4 IKAROS family zinc finger 4 (Eos ) Nucleus transcription regulator IL10 interleukin 10 Extracellular Space cytokine IL17B interleukin 17B Extracellular Space cytokine IL18 ( includes interleukin 18 ( interferon - gamma- inducing factor ) Extracellular Space cytokine EG : 16173 ) b IL1B interleukin 1 , beta Extracellular Space cytokine ILIRAPL1 interleukin 1 receptor accessory protein - like 1 Plasma Membrane transmembrane receptor ILIRN interleukin 1 receptor antagonist Extracellular Space cytokine IL2 interleukin 2 Extracellular Space cytokine IL3 interleukin 3 ( colony- stimulating factor ,multiple ) Extracellular Space other IL31 interleukin 31 unknown other IL6 interleukin 6 ( interferon , beta 2 ) Extracellular Space cytokine IMPG1 interphotoreceptor matrix proteoglycan 1 Extracellular Space other INGX inhibitor of growth family , X - linked , pseudogene unknown other INPP5E inositolpolyphosphate - 5 -phosphatase , 72 kDa Cytoplasm phosphatase INPP5K inositol polyphosphate - 5 -phosphatase K Cytoplasm phosphatase INSL5 insulin - like 5 Extracellular Spaceull, other US 9 ,944 ,986 B2 43 44 TABLE 1 - continued Rapamycin -sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type ( s ) IQCF1 IQ motif containing F1 unknown other IRS2 insulin receptor substrate 2 Cytoplasm enzyme IRX4 iroquois homeobox 4 Nucleus transcription regulator ISX intestine - specific homeobox unknown other ITGB3 integrin , beta 3 (platelet glycoprotein IIIa , antigen Plasma Membrane transmembrane CD61) receptor ITGBL1 integrin , beta - like 1 (with EGF- like repeat domains) unknown other JAK1 ( includes Janus kinase 1 Cytoplasm kinase EG : 16451 ) JAKMIP3 Janus kinase and microtubule interacting protein 3 unknown other JARID2 jumonji , AT rich interactive domain 2 Nucleus transcription regulator JUN jun proto -oncogene Nucleus transcription regulator KANK2 KN motif and ankyrin repeat domains 2 Nucleus transcription regulator KCNA3 potassium voltage -gated channel, shaker- related Plasma Membrane ion channel subfamily, member 3 KCNA4 potassium voltage - gated channel, shaker- related Plasma Membrane ion channel subfamily, member 4 KCNAZ potassium voltage- gated channel , shaker - related Plasma Membrane ion channel subfamily , member 7 KCNAB1 potassium voltage- gated channel, shaker - related Plasma Membrane ion channel subfamily , beta member 1 KCNB2 potassium voltage- gated channel, Shab - related Plasma Membrane ion channel subfamily, member 2 KCNC1 potassium voltage - gated channel , Shaw - related Plasma Membrane ion channel subfamily , member 1 KCNC2 potassium voltage - gated channel, Shaw - related Plasma Membrane ion channel subfamily , member 2 KCND3 potassium voltage - gated channel, Shal- related Plasma Membrane ion channel subfamily, member 3 KCNG4 potassium voltage- gated channel, subfamily G , Plasma Membrane ion channel member 4 KCNH7 potassium voltage - gated channel, subfamily H (eag Plasma Membrane ion channel related ) , member 7 KCNIP2 Kv channel interacting protein 2 Cytoplasm other KCNJ5 potassium inwardly - rectifying channel , subfamily J , Plasma Membrane ion channel member 5 KCNK10 potassium channel , subfamily K , member 10 Plasma Membrane ion channel KCNK17 potassium channel , subfamily K , member 17 Plasma Membrane ion channel KCNQ1 potassium voltage - gated channel, KQT- like Plasma Membrane ion channel subfamily , member 1 KCNQ1DN KCNQ1 downstream neighbor (non -protein coding ) unknown other KCP ( includes kielin / chordin - like protein Extracellular Space other EG : 296952 ) KCTD19 potassium channel tetramerisation domain containing unknown other 19 KCTD4 potassium channel tetramerisation domain containing 4 unknown ion channel KIAA0355 KIAA0355 unknown other KIAA0825 KIAA0825 unknown other KIAA1045 KIAA1045 unknown other KIAA1109 KIAA1109 unknown other KIAA1239 KIAA1239 unknown other KIAA1407 KIAA1407 unknown other KIAA1462 KIAA1462 unknown other KIAA1522 KIAA1522 unknown other KIAA1683 KIAA1683 Cytoplasm other KIF6 kinesin family member 6 Nucleus other KIRREL3 kin of IRRE like 3 (Drosophila ) Extracellular Space other KL klotho Extracellular Space enzyme KLF12 Kruppel - like factor 12 Nucleus transcription regulator KLF2 Kruppel- like factor 2 (lung ) Nucleus transcription regulator KLHDC9 kelch domain containing 9 unknown other KLHL24 kelch - like 24 (Drosophila ) unknown other KLHL4 kelch - like 4 (Drosophila ) Cytoplasm other KLRB1 killer cell lectin - like receptor subfamily B , member 1 Plasma Membrane transmembrane receptor KRT2 keratin 2 Cytoplasm other KRT6B keratin 6B Cytoplasm other KRT72 keratin 72 unknown other KRT75 keratin 75 Cytoplasm other KRT82 keratin 82 Cytoplasm other US 9 ,944 ,986 B2 45 46 TABLE 1 - continued Rapamycin - sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type( s ) KRTAP1 - 1 keratin associated protein 1 - 1 unknown other KRTAP1- 3 keratin associated protein 1 - 3 unknown other KRTAP15 - 1 keratin associated protein 15 - 1 unknown other KRTAP4 - 7 keratin associated protein 4 - 7 unknown other KRTAP9 - 2 keratin associated protein 9 - 2 unknown other LAIR2 leukocyte - associated immunoglobulin - like receptor 2 Plasma Membrane other LAMA1 laminin , alpha 1 Extracellular Space other LAYN layilin Plasma Membrane other LCE1E late cornified envelope 1E unknown other LECT1 leukocyte cell derived chemotaxin 1 Extracellular Space other LEF1 lymphoid enhancer -binding factor 1 Nucleus transcription regulator LEFTY1 left - right determination factor 1 Extracellular Space growth factor LEMD1 LEM domain containing 1 unknown other LEP leptin Extracellular Space growth factor LGALS13 lectin , galactoside- binding , soluble, 13 unknown enzyme LGALS2 lectin , galactoside- binding , soluble , 2 Cytoplasm other LGR5 leucine- rich repeat containing G protein - coupled Plasma Membrane G -protein receptor 5 coupled receptor LIFR leukemia inhibitory factor receptor alpha Plasma Membrane transmembrane receptor LILRB2 leukocyte immunoglobulin - like receptor, subfamily B Plasma Membrane other (with TM and ITIM domains) , member 2 LINC00317 long intergenic non -protein coding RNA 317 unknown other LINC00477 long intergenic non -protein coding RNA 477 unknown other LIPF lipase , gastric Extracellular Space enzyme LOC100128098 uncharacterized LOC100128098 unknown other LOC100128108 uncharacterized LOC100128108 unknown other LOC100129406 uncharacterized LOC100129406 unknown other LOC100129476 uncharacterized LOC100129476 unknown other LOC100129775 uncharacterized LOC100129775 unknown other LOC100130278 uncharacterized LOC100130278 unknown other LOC100130776 uncharacterized LOC100130776 unknown other LOC100130815 uncharacterized LOC100130815 unknown other LOC100131176 uncharacterized LOC100131176 unknown other LOC100132116 uncharacterized LOC100132116 unknown other LOC100132363 uncharacterized LOC100132363 unknown other LOC100170939 glucuronidase , beta pseudogene unknown other LOC100190938 uncharacterized LOC100190938 unknown other LOC100190986 uncharacterized LOC100190986 unknown other LOC100288966 /POTED POTE ankyrin domain family ,member D Plasma Membrane other LOC120824 / SPRYD5 SPRY domain containing 5 unknown other LOC283174 uncharacterized LOC283174 unknown other LOC283663 uncharacterized LOC283663 unknown other LOC283665 uncharacterized LOC283665 unknown other LOC284260 uncharacterized LOC284260 unknown other LOC284861 uncharacterized LOC284861 unknown other LOC286071 uncharacterized LOC286071 unknown other LOC286382 uncharacterized LOC286382 unknown other LOC339260 uncharacterized LOC339260 unknown other LOC389023 uncharacterized LOC389023 unknown other LOC389043 uncharacterized LOC389043 unknown other LOC390705 protein phosphatase 2 , regulatory subunit B " , beta unknown other pseudogene LOC400620 uncharacterized LOC400620 unknown other LOC400655 uncharacterized LOC400655 unknown other LOC401317 uncharacterized LOC401317 unknown other LOC441601 septin 7 pseudogene unknown other LOC474358 uncharacterized BC042079 locus unknown other LOC644192 uncharacterized LOC644192 unknown other LOC646471 uncharacterized LOC646471 unknown other LOC646627 phospholipase inhibitor unknown other LOC647107 uncharacterized LOC647107 unknown other LOC647946 uncharacterized LOC647946 unknown other LOC728093 / LOC729915 putative POM121- like protein 1 unknown other LOC728323 uncharacterized LOC728323 unknown other LOC729121 uncharacterized LOC729121 unknown other LOC729970 hCG2028352 - like unknown other LOX ( includes lysyl oxidase Extracellular Space enzyme EG : 16948 ) LOXL3 lysyl oxidase - like 3 Extracellular Space enzyme LOXL4 lysyl oxidase - like 4 Extracellular Space enzyme LPIN2 lipin 2 Nucleus phosphatase LRCH2 leucine- rich repeats and calponin homology (CH ) unknown other domain containing 2 US 9 ,944 ,986 B2 47 48 TABLE 1 - continued Rapamycin - sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type ( s) LRP1 ( includes low density lipoprotein receptor - related protein 1 Plasma Membrane transmembrane EG : 16971 ) receptor LRP1B low density lipoprotein receptor- related protein 1B Plasma Membrane transmembrane receptor LRRC27 leucine rich repeat containing 27 unknown other LRRC37A3 ( includes leucine rich repeat containing 37 , member A3 unknown other others ) LRRC48 leucine rich repeat containing 48 Cytoplasm other LRRC71 leucine rich repeat containing 71 unknown other LRRCC1 leucine rich repeat and coiled -coil domain containing 1 Nucleus transporter LTBP1 latent transforming growth factor beta binding protein 1 Extracellular Space other LUM lumican Extracellular Space other LYPD2 LY6 /PLAUR domain containing 2 unknown other LYPD6 LY6 /PLAUR domain containing 6 Extracellular Space other MACF1 microtubule - actin crosslinking factor 1 Cytoplasm enzyme MAEL maelstrom homolog ( Drosophila ) Cytoplasm other MAL mal , T - cell differentiation protein Plasma Membrane transporter MAPK4 mitogen -activated protein kinase 4 Cytoplasm kinase MAPT microtubule - associated protein tau Cytoplasm other MARK1 MAP /microtubule affinity - regulating kinase 1 Cytoplasm kinase MARVELD3 MARVEL domain containing 3 unknown other MAS1 MASI oncogene Plasma Membrane G -protein coupled receptor MBD3L2 ( includes methyl- CpG binding domain protein 3 -like 2 unknown other others ) MBP myelin basic protein Extracellular Space other MCHR2 melanin - concentrating hormone receptor 2 Plasma Membrane G -protein coupled receptor ????1 multiple C2 domains, transmembrane 1 unknown other MECOM MDS1 and EVII complex locus Nucleus transcription regulator MEG3 maternally expressed 3 (non - protein coding ) unknown other METTL21A methyltransferase like 21A unknown other METTL7A methyltransferase like 7A unknown other MFAP5 microfibrillar associated protein 5 Extracellular Space other MGC24103 uncharacterized MGC24103 unknown other MGC39545 uncharacterized LOC403312 unknown other MGC70870 C - terminal binding protein 2 pseudogene unknown other MGEA5 meningioma expressed antigen 5 (hyaluronidase ) Cytoplasm enzyme MIA2 melanoma inhibitory activity 2 Extracellular Space other MIER1 mesoderm induction early response 1 homolog Nucleus other ( Xenopus laevis ) MIR7- 3HG MIR7- 3 host gene (non -protein coding ) unknown other MIS18BP1 MIS18 binding protein 1 Nucleus other MLF1 myeloid leukemia factor 1 Nucleus other MLL myeloid /lymphoid or mixed - lineage leukemia Nucleus transcription ( trithorax homolog, Drosophila ) regulator MLLT4 myeloid / lymphoid or mixed - lineage leukemia Nucleus other ( trithorax homolog , Drosophila ); translocated to , 4 MME membrane metallo -endopeptidase Plasma Membrane peptidase MMP12 matrix metallopeptidase 12 (macrophage elastase ) Extracellular Space peptidase MMP16 matrix metallopeptidase 16 (membrane - inserted ) Extracellular Space peptidase MMP2 matrix metallopeptidase 2 (gelatinase A , 72 kDa Extracellular Space peptidase gelatinase , 72 kDa type IV collagenase ) MMP8 matrix metallopeptidase 8 (neutrophil collagenase ) Extracellular Space peptidase MMP9 matrix metallopeptidase 9 ( gelatinase B , 92 kDa Extracellular Space peptidase gelatinase , 92 kDa type IV collagenase ) MNT MAX binding protein Nucleus transcription regulator MORNS MORN repeat containing 5 unknown other MOXD1 monooxygenase, DBH - like 1 Cytoplasm enzyme MPP4 membrane protein , palmitoylated 4 (MAGUK p55 Cytoplasm kinase subfamily member 4 ) MPPED2 metallophosphoesterase domain containing 2 unknown other MRO maestro Nucleus other MSMB microseminoprotein , beta Extracellular Space other MTHFR methylenetetrahydrofolate reductase (NAD (P ) H ) Cytoplasm enzyme MTL5 metallothionein - like 5 , testis -specific ( tesmin ) Cytoplasm other MTMR7 myotubularin related protein 7 Cytoplasm phosphatase MUC17 mucin 17 , cell surface associated Plasma Membrane other MXI1 MAX interactor 1 Nucleus transcription regulator US 9 ,944 ,986 B2 49 50 TABLE 1 - continued Rapamycin- sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type( s ) MYCL1 v -myc myelocytomatosis viral oncogene homolog 1 , Nucleus transcription lung carcinoma derived ( avian ) regulator MYF6 myogenic factor 6 (herculin ) Nucleus wtranscription regulator MYO16 myosin XVI Cytoplasm other ????? myosin IIIB unknown kinase MYOM1 myomesin 1, 185 kDa Cytoplasm other MYOZ1 myozenin 1 Cytoplasm other MYPN myopalladin Cytoplasm other N4BP2L1 NEDD4 binding protein 2 - like 1 unknown other NAG20 NAG20 unknown other NAV1 neuron navigator 1 Cytoplasm enzyme NAV2 neuron navigator 2 Nucleus other NBR1 neighbor of BRCA1 gene 1 Cytoplasm other NDRG2 NDRG family member 2 Cytoplasm other NECAB2 N - terminal EF -hand calcium binding protein 2 Cytoplasm other NEGR1 neuronal growth regulator 1 Extracellular Space other NEUROD4 neuronal differentiation 4 Nucleus other NEXN - AS1 NEXN antisense RNA 1 (non - protein coding) unknown other NFIB nuclear factor I / B Nucleus transcription regulator NIPBL Nipped - B homolog ( Drosophila ) Nucleus transcription regulator NKX3 - 2 NK3 homeobox 2 Nucleus transcription regulator NLRP13 NLR family , pyrin domain containing 13 unknown other NMNAT2 nicotinamide nucleotide adenylyltransferase 2 Cytoplasm enzyme NMU neuromedin U Extracellular Space other NOTCH2NL notch 2 N - terminal like unknown other NOX1 NADPH oxidase 1 Cytoplasm ion channel NPTX1 neuronal pentraxin I Extracellular Space other NR2F2 nuclear receptor subfamily 2 , group F , member 2 Nucleus ligand dependent nuclear receptor NR4A1 nuclear receptor subfamily 4 , group A , member 1 Nucleus ligand dependent nuclear receptor NR4A2 nuclear receptor subfamily 4 , group A , member 2 Nucleus ligand dependent nuclear receptor NRG2 neuregulin 2 Extracellular Space growth factor NRIP2 nuclear receptor interacting protein 2 Nucleus transcription regulator NRXN3 neurexin 3 Plasma Membrane transporter NSUNT NOP2 / Sun domain family ,member 7 unknown other NTN4 netrin 4 Extracellular Space other NTSR1 neurotensin receptor 1 (high affinity ) Plasma Membrane G -protein coupled receptor NUDT9P1 nudix (nucleoside diphosphate linked moiety X ) - type unknown other motif 9 pseudogene 1 NUP210P1 nucleoporin 210 kDa pseudogene 1 unknown other NYAP1 neuronal tyrosine -phosphorylated phosphoinositide unknown other 3 - kinase adaptor 1 OCA2 ( includes oculocutaneous albinism II Plasma Membrane transporter EG : 18431) ODF1 outer dense fiber of sperm tails 1 Cytoplasm other OGN osteoglycin Extracellular Space growth factor OIT3 oncoprotein induced transcript 3 Nucleus other OLFML2A olfactomedin - like 2A Extracellular Spacealiitwaliohudhudihukalului other OLIG2 oligodendrocyte lineage transcription factor 2 Nucleus transcription regulator OPRM1 opioid receptor, mu 1 Plasma Membrane G -protein coupled receptor OR10J1 olfactory receptor, family 10 , subfamily J, member 1 Plasma Membrane G -protein coupled receptor OR10T2 olfactory receptor, family 10 , subfamily T, member 2 Plasma Membrane other OR1E1 olfactory receptor , family 1, subfamily E ,member 1 Plasma Membrane G - protein coupled receptor US 9 ,944 ,986 B2 5151 TABLE 1 - continued Rapamycin - sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type( s ) OR2C3 olfactory receptor, family 2 , subfamily C , member 3 Plasma Membrane other OR2L13 olfactory receptor, family 2 , subfamily L , member 13 Plasma Membrane G - protein coupled receptor OR2S2 olfactory receptor, family 2, subfamily S , member 2 Plasma Membrane G - protein coupled receptor OR2V2 olfactory receptor, family 2, subfamily V ,member 2 Plasma Membrane G -protein coupled receptor OR4N4 olfactory receptor , family 4 , subfamily N ,member 4 Plasma Membrane G - protein coupled receptor OR51E1 olfactory receptor, family 51 , subfamily E , member 1 Plasma Membrane G -protein coupled receptor OR51E2 olfactory receptor, family 51, subfamily E , member 2 Plasma Membrane G -protein coupled receptor OR5101 olfactory receptor , family 51, subfamily Q , member 1 Plasma Membrane G - protein coupled receptor OR52B2 olfactory receptor, family 52, subfamily B , member 2 Plasma Membrane G - protein coupled receptor OR5AK2 olfactory receptor, family 5 , subfamily AK , member 2 Plasma Membrane G -protein coupled receptor OSBPL2 oxysterol binding protein - like 2 Cytoplasm other OSOSTalpha organic solute transporter alpha Plasma Membrane transporter OTX1 orthodenticle homeobox 1 Nucleus transcription regulator OVOL1 ovo - like 1 ( Drosophila ) Nucleus transcription regulator P2RY13 purinergic receptor P2Y, G -protein coupled , 13 Plasma Membrane G -protein coupled receptor P2RY14 purinergic receptor P2Y , G -protein coupled , 14 Plasma Membrane G - protein coupled receptor PABPC5 poly ( A ) binding protein , cytoplasmic 5 Cytoplasm other PAOX polyamine oxidase (exo -N4 - amino ) Cytoplasm enzyme PARVA parvin , alpha Cytoplasm other PASD1 PAS domain containing 1 Nucleus other PAX3 paired box 3 Nucleus transcription regulator PCDH11X /PCDH11Y protocadherin 11 Y - linked Plasma Membrane other PCDH18 protocadherin 18 Extracellular Space other PCDH7 protocadherin 7 Plasma Membrane other PCDHB10 protocadherin beta 10 Plasma Membrane Hinduiwother PCDHB5 protocadherin beta 5 Plasma Membrane other PCDHGA9 protocadherin gamma subfamily A , 9 unknown other PCDHGBP protocadherin gamma subfamily B , 8 pseudogene unknown other PCMTD2 protein - L - isoaspartate ( D - aspartate ) O Cytoplasm enzyme methyltransferase domain containing 2 PCNXL2 pecanex - like 2 (Drosophila ) unknown other PCSK2 proprotein convertase subtilisin /kexin type 2 Extracellular Space peptidase PDC phosducin Cytoplasm other PDCD4 programmed cell death 4 (neoplastic transformation Nucleus other inhibitor) PDE1A phosphodiesterase 1A , calmodulin - dependent Cytoplasm enzyme PDE4C phosphodiesterase 4C , CAMP- specific Cytoplasm enzyme PDE4DIP phosphodiesterase 4D interacting protein Cytoplasm enzyme PDEGA phosphodiesterase 6A , CGMP- specific , rod , alpha Plasma Membrane enzyme PDESB phosphodiesterase 8B Cytoplasm enzyme PDK4 pyruvate dehydrogenase kinase , isozyme 4 Cytoplasm kinase PDLIM3 PDZ and LIM domain 3 Cytoplasm llwallelother PDZD9 PDZ domain containing 9 unknown other PDZRN4 PDZ domain containing ring finger 4 unknown other PES1 pescadillo ribosomal biogenesis factor 1 Nucleus other PEX5L peroxisomal biogenesis factor 5 - like Cytoplasm ion channel PHACTR1 phosphatase and actin regulator 1 Cytoplasm other PHF12 PHD finger protein 12 Nucleus transcription regulator PHLDA3 pleckstrin homology - like domain , family A , member 3 Plasma Membrane other PHLDB2 pleckstrin homology - like domain , family B , member 2 Cytoplasm other US 9 ,944 ,986 B2 53 54 TABLE 1 - continued Rapamycin -sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type( s ) PHYHD1 phytanoyl- CoA dioxygenase domain containing 1 unknown other PHYHIPL phytanoyl- CoA 2 - hydroxylase interacting protein - like Cytoplasm other PIM1 pim - 1 oncogene Cytoplasm kinase PIP prolactin - induced protein Extracellular Space other PITPNM2 phosphatidylinositol transfer protein , membrane Cytoplasm enzyme associated 2 PKD1L1 polycystic kidney disease 1 like 1 Extracellular Space other PKHD1 polycystic kidney and hepatic disease 1 ( autosomal Plasma Membrane other recessive ) PKP1 plakophilin 1 ( ectodermal dysplasia / skin fragility Plasma Membrane other syndrome ) PLA2R1 phospholipase A2 receptor 1 , 180 kDa Plasma Membrane transmembrane receptor PLAT plasminogen activator, tissue Extracellular Space peptidase PLB1 ( includes phospholipase B1 Cytoplasm enzyme EG : 151056 ) PLCD4 phospholipase C , delta 4 Cytoplasm enzyme PLCH1 phospholipase C , eta 1 Cytoplasm enzyme PLCZ1 phospholipase C , zeta 1 unknown enzyme PLD1 phospholipase D1, phosphatidylcholine - specific Cytoplasm enzyme PLD3 phospholipase D family , member 3 Cytoplasm enzyme PLEKHA6 pleckstrin homology domain containing , family A unknown Withother member 6 PLEKHH2 pleckstrin homology domain containing , family H Cytoplasm other ( with MyTH4 domain ) member 2 PLGLB1/ PLGLB2 plasminogen - like B2 Extracellular Space peptidase PLN phospholamban Cytoplasm other PLSCR2 phospholipid scramblase 2 unknown other PLSCR4 phospholipid scramblase 4 Plasma Membrane enzyme PM20D1 peptidase M20 domain containing 1 unknown peptidase PMCH pro -melanin -concentrating hormone Extracellular Space other PMCHL1 pro -melanin -concentrating hormone- like 1 , Extracellular Space other pseudogene PNLIP pancreatic lipase Extracellular Space enzyme PNPLA1 patatin - like phospholipase domain containing 1 unknown enzyme PNRC1 proline - rich nuclear receptor coactivator 1 Nucleus other POLK polymerase (DNA directed ) kappa Nucleus enzyme POLR2M polymerase ( RNA ) II (DNA directed ) polypeptide M Nucleus other PON3 paraoxonase 3 Extracellular Space enzyme POTEE /POTEF POTE ankyrin domain family , member F unknown other POU4F2 POU class 4 homeobox 2 Nucleus transcription regulator PPAPDC1A phosphatidic acid phosphatase type 2 domain unknown phosphatase containing 1A PPIC peptidylprolyl isomerase C ( cyclophilin C ) Cytoplasm enzyme PRICKLE2 prickle homolog 2 (Drosophila ) Nucleus other PRKAA2 protein kinase , AMP- activated , alpha 2 catalytic Cytoplasm kinase subunit PRKG1 protein kinase , cGMP- dependent , type I Cytoplasm kinase PRODH2 proline dehydrogenase (oxidase ) 2 Cytoplasm enzyme PRR16 proline rich 16 unknown other PRSS23 protease , serine , 23 Extracellular Space peptidase PSORS1C1 psoriasis susceptibility 1 candidate 1 unknown thehellbuahlilinilbiliother PTCH1 patched 1 Plasma Membrane transmembrane receptor PTGER3 prostaglandin E receptor 3 (subtype EP3) Plasma Membrane G -protein coupled receptor PTGS2 prostaglandin - endoperoxide synthase 2 Cytoplasm enzyme (prostaglandin G /H synthase and cyclooxygenase ) PTPRC protein tyrosine phosphatase , receptor type , C Plasma Membrane phosphatase PTPRZ1 protein tyrosine phosphatase, receptor- type , Z Plasma Membrane phosphatase polypeptide 1 PYCR1 pyrroline - 5 - carboxylate reductase 1 Cytoplasm enzyme PZP pregnancy - zone protein Extracellular Space other RAB37 RAB37 , member RAS oncogene family Cytoplasm enzyme RAB39A RAB39A , member RAS oncogene family Cytoplasm enzyme RAB3C ( includes RAB3C ,member RAS oncogene family Cytoplasm enzyme EG : 115827 ) RAETIE retinoic acid early transcript 1E Plasma Membrane other RAG1 recombination activating gene 1 Nucleus enzyme RAMP1 receptor (G protein - coupled ) activity modifying Plasma Membrane transporter protein 1 RAVER2 ribonucleoprotein , PTB -binding 2 Nucleus other RBMY1A1 ( includes RNA binding motif protein , Y - linked , family 1 , Nucleus other others ) member A1 US 9 ,944 ,986 B2 55 56 TABLE 1 - continued Rapamycin- sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type( s ) RBPMS2 RNA binding protein with multiple splicing 2 unknown other RERGL RERG /RAS - like unknown other REST RE1 - silencing transcription factor Nucleus transcription regulator RET ret proto - oncogene Plasma Membrane kinase REXO1Li ( includes REX1 , RNA exonuclease 1 homolog ( S . cerevisiae ) unknown enzyme others ) like 1 RGNEF 190 kDa guanine nucleotide exchange factor Cytoplasm other RHO rhodopsin Plasma Membrane G - protein coupled receptor RHOT1 ras homolog family member T1 Cytoplasm enzyme RIMBP3 ( includes RIMS binding protein 3 Nucleus other others ) RIMS2 regulating synaptic membrane exocytosis 2 unknown other RIN2 Ras and Rab interactor 2 Cytoplasm other RIT2 Ras - like without CAAX 2 Plasma Membrane enzyme RMI2 RMI2 , RecQ mediated genome instability 2 , homolog unknown other ( S . cerevisiae ) RND3 Rho family GTPase 3 Cytoplasm enzyme RNF125 ring finger protein 125 , E3 ubiquitin protein ligase unknown other RNF128 ring finger protein 128 , E3 ubiquitin protein ligase Cytoplasm enzyme RNF133 ring finger protein 133 Cytoplasm other RNF175 ring finger protein 175 unknown other RNF8 ring finger protein 8, E3 ubiquitin protein ligase Nucleus enzyme RORB RAR - related orphan receptor B Nucleus ligand dependent nuclear receptor RPL32P3 ribosomal protein L32 pseudogene 3 unknown other RPS6KB1 ribosomal protein S6 kinase , 70 kDa, polypeptide 1 Cytoplasm kinase RRAD Ras- related associated with diabetes Cytoplasm enzyme RUFY2 RUN and FYVE domain contain Nucleus other RUNX1T1 runt - related transcription factor 1 ; translocated to , 1 Nucleus transcription (cyclin D - related ) regulator RUNX2 runt- related transcription factor 2 Nucleus transcription regulator SALL3 sal - like 3 (Drosophila ) Nucleus other SCARNA17 small Cajal body - specific RNA 17 unknown other SCGN secretagogin , EF -hand calcium binding protein Cytoplasm other SCN3B sodium channel, voltage - gated , type III , beta subunit Plasma Membrane ion channel SCN8A sodium channel, voltage gated , type VIII , alpha Plasma Membrane buitituluion channel subunit SEMA4C sema domain , immunoglobulin domain (Ig ) , Plasma Membrane other transmembrane domain ( TM ) and short cytoplasmic domain , ( semaphorin ) 4C SEMASA sema domain , seven thrombospondin repeats ( type 1 Plasma Membrane transmembrane and type 1 - like ), transmembrane domain ( TM ) and receptor short cytoplasmic domain , ( semaphorin ) 5A SEMA6A sema domain , transmembrane domain ( TM ) , and Plasma Membrane other cytoplasmic domain , ( semaphorin ) 6A SEMG2 semenogelin II Extracellular Space other SERPINA10 serpin peptidase inhibitor, clade A ( alpha - 1 Extracellular Space Other antiproteinase , antitrypsin ), member 10 SERPINB3 serpin peptidase inhibitor , clade B ( ovalbumin ) , Extracellular Space other member 3 SERPINB4 serpin peptidase inhibitor , clade B ( ovalbumin ), Cytoplasm other member 4 SERPINDI serpin peptidase inhibitor , clade D (heparin cofactor) , Extracellular Space other member 1 SERPINE1 serpin peptidase inhibitor , clade E (nexin , Extracellular Space other plasminogen activator inhibitor type 1 ) ,member 1 SERTAD4 SERTA domain containing 4 unknown other SGCD sarcoglycan , delta ( 35 kDa dystrophin - associated Cytoplasm other glycoprotein ) SGCZ sarcoglycan , zeta Plasma Membrane other SGTA small glutamine- rich tetratricopeptide repeat ( TPR ) Cytoplasm other containing , alpha SH3PXD2A SH3 and PX domains 2A Cytoplasm other SHANK3 SH3 and multiple ankyrin repeat domains 3 Cytoplasm transcription regulator SHROOM2 shroom family member 2 Plasma Membrane ion channel SHROOM4 shroom family member 4 Plasma Membrane other SIGLEC1 sialic acid binding Ig - like lectin 1 , sialoadhesin Plasma Membrane other SIM2 single - minded homolog 2 ( Drosophila ) Nucleus transcription regulator US 9 ,944 ,986 B2 57 58 TABLE 1 - continued Rapamycin - sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type ( s) SLC13A1 solute carrier family 13 (sodium / sulfate symporters ), Plasma Membrane transporter member 1 SLC15A1 solute carrier family 15 (oligopeptide transporter ), Plasma Membrane transporter member 1 SLC16A5 solute carrier family 16 , member 5 (monocarboxylic Plasma Membrane transporter acid transporter 6 ) SLC18A1 solute carrier family 18 (vesicular monoamine ), Plasma Membrane transporter member 1 SLC18A3 solute carrier family 18 (vesicular acetylcholine ) , Plasma Membrane transporter member 3 SLC19A3 solute carrier family 19 , member 3 Plasma Membrane transporter SLC22A18 solute carrier family 22 , member 18 Plasma Membrane transporter SLC22A9 solute carrier family 22 (organic anion transporter ) , Plasma Membrane transporter member 9 SLC25A27 solute carrier family 25 , member 27 Cytoplasm transporter SLC25A36 solute carrier family 25 , member 36 Cytoplasm transporter SLC35F3 solute carrier family 35 , member F3 unknown other SLC38A3 solute carrier family 38 , member 3 Plasma Membrane transporter SLC46A2 solute carrier family 46 , member 2 Plasma Membrane transporter SLC4A2 solute carrier family 4 , anion exchanger, member 2 Plasma Membrane transporter (erythrocyte membrane protein band 3 - like 1 ) SLC6A1 solute carrier family 6 ( neurotransmitter transporter , Plasma Membrane transporter GABA ) , member 1 SLC6A11 solute carrier family 6 (neurotransmitter transporter, Plasma Membrane transporter GABA ) , member 11 SLC6A15 solute carrier family 6 (neutral amino acid Plasma Membrane transporter transporter ), member 15 SLC6A19 solute carrier family 6 (neutral amino acid Plasma Membrane transporter transporter ), member 19 SLC9B1 solute carrier family 9 , subfamily B (NHA1 , cation Plasma Membrane other proton antiporter 1 ) , member 1 SLCO1A2 solute carrier organic anion transporter family , Plasma Membrane transporter member 1A2 SLFN5 schlafen family member 5 Nucleus enzyme SLITRK1 SLIT and NTRK - like family , member 1 unknown other SLITRK5 SLIT and NTRK - like family , member 5 unknown other SMA4 glucuronidase , beta pseudogene unknown other SMAD3 SMAD family member 3 Nucleus transcription regulator SMG1 smg- 1 homolog, phosphatidylinositol 3 - kinase - related Cytoplasm kinase kinase ( C . elegans) SMOC1 SPARC related modular calcium binding 1 Extracellular Space other SNTB1 syntrophin , beta 1 (dystrophin - associated protein Al , Plasma Membrane other 59 kDa , basic component 1 ) SOCS2 suppressor of cytokine signaling 2 Cytoplasm other SORBS2 sorbin and SH3 domain containing 2 Plasma Membrane other SOX11 SRY ( sex determining region Y ) -box 11 Nucleus transcription regulator SOX21 SRY (sex determining region Y ) - box 21 Nucleus transcription regulator SOX7 SRY ( sex determining region Y ) -box 7 Nucleus transcription regulator SP100 SP100 nuclear antigen Nucleus transcription regulator SPATA17 spermatogenesis associated 17 unknown other SPDYA speedy homolog A (Xenopus laevis ) Nucleus other SPINLW1 serine peptidase inhibitor- like, with Kunitz and WAP Extracellular Space other domains 1 ( eppin ) SPOCK1 sparc /osteonectin , cwcv and kazal- like domains Extracellular Space other proteoglycan (testican ) 1 SPP1 ( includes secreted phosphoprotein 1 Extracellular Space cytokine EG : 20750 ) SPRED2 sprouty - related , EVH1 domain containing 2 Extracellular Space cytokine SPSB3 splA / ryanodine receptor domain and SOCS box unknown other containing 3 SPTB spectrin , beta , erythrocytic Plasma Membrane other SSBP1 single - stranded DNA binding protein 1 Cytoplasm other SSPO SCO - spondin homolog (Bos taurus) Cytoplasm other SSTR1 somatostatin receptor 1 Plasma Membrane G - protein coupled receptor SSX4 / SSX4B synovial sarcoma , X breakpoint 4 Nucleus other SSX8 synovial sarcoma , X breakpoint 8 unknown other STUGAL6 ST3 beta - galactoside alpha - 2 , 3 - sialyltransferase 6 Cytoplasm enzyme US 9 ,944 ,986 B2 59 TABLE 1 - continued Rapamycin - sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type( s ) STOGAL2 ST6 beta - galactosamide alpha - 2 , 6 - sialyltranferase 2 Cytoplasm enzyme STOGALNAC2 ST6 (alpha - N - acetyl- neuraminyl - 2 , 3 -beta - galactosyl Cytoplasm enzyme 1 , 3 )- N -acetylgalactosaminide alpha - 2 , 6 sialyltransferase 2 STAG3L1 stromal antigen 3 - like 1 unknown other STARD13 STAR -related lipid transfer ( START) domain Cytoplasm other containing 13 STK31 serine /threonine kinase 31 Cytoplasm kinase STK32B serine /threonine kinase 32B unknown kinase STMN1 stathmin 1 Cytoplasm other STMN3 stathmin - like 3 Nucleus other STON1- GTF2AIL STON1- GTF2AIL readthrough Nucleus transcription regulator STRAS stimulated by retinoic acid gene 8 homolog (mouse ) unknown other STS steroid sulfatase (microsomal ) , isozyme S Cytoplasm enzyme STX1B syntaxin 1B Plasma Membrane ion channel SYCE2 synaptonemal complex central element protein 2 Nucleus other SYCP3 synaptonemal complex protein 3 Nucleus other SYNE2 spectrin repeat containing , nuclear envelope 2 Nucleus other SYNPO2 synaptopodin 2 Cytoplasm other SYT14 synaptotagmin XIV unknown transporter TAC1 tachykinin , precursor 1 Extracellular Space other TACSTD2 tumor- associated calcium signal transducer 2 Plasma Membrane other TAKR aldo -keto reductase , truncated unknown enzyme TAOK1 TAO kinase 1 Cytoplasm kinase TAS2R42 taste receptor, type 2 , member 42 unknown other TBX4 T -box 4 Nucleus transcription regulator TCEA3 transcription elongation factor A (SII ), 3 Nucleus transcription regulator TCF12 transcription factor 12 Nucleus transcription regulator TCLIB T -cell leukemia / lymphoma 1B unknown other TCTEX1D1 Tetexl domain containing 1 unknown other TDH L - threonine dehydrogenase Cytoplasm enzyme TEDDM1 transmembrane epididymal protein 1 unknown other TFAP2A transcription factor AP- 2 alpha (activating enhancer Nucleus transcription binding protein 2 alpha ) regulator TFAP2D transcription factor AP - 2 delta (activating enhancer Nucleus transcription binding protein 2 delta ) regulator TGFB3 transforming growth factor, beta 3 Extracellular Space growth factor TGM4 transglutaminase 4 (prostate ) Extracellular Space enzyme THBS1 thrombospondin 1 Extracellular Space other ???? thrombopoietin Extracellular Space cytokine THRA ( includes thyroid hormone receptor, alpha Nucleus ligand EG : 21833 ) dependent nuclear receptor THSD7B thrombospondin , type I, domain containing 7B unknown other TIMM17B translocase of inner mitochondrial membrane 17 Cytoplasm transporter homolog B (yeast ) TIMP2 ( includes TIMPmetallopeptidase inhibitor 2 Extracellular Space other EG : 21858 ) TINAG tubulointerstitial nephritis antigen Extracellular Space peptidase TLL1 tolloid - like 1 Extracellular Space peptidase TLR4 toll- like receptor 4 Plasma Membrane transmembrane receptor TLX1 T - cell leukemia homeobox 1 Nucleus transcription regulator TM4SF18 transmembrane 4 L six family member 18 unknown other TM4SF4 transmembrane 4 L six family member 4 Plasma Membrane other ???? transmembrane channel- like 3 unknown other TMEM192 transmembrane protein 192 unknown other TMEM37 transmembrane protein 37 Plasma Membrane ion channel TMEM45B transmembrane protein 45B Extracellular Space other TMEM47 transmembrane protein 47 Plasma Membrane other TMEM56 transmembrane protein 56 unknown other TMPRSS11A transmembrane protease , serine 11A unknown peptidase TNFAIP6 tumor necrosis factor, alpha - induced protein 6 Extracellular Space other TNFRSF10C tumor necrosis factor receptor superfamily , member Plasma Membrane transmembrane 10c, decoy without an intracellular domain receptor TNFRSF19 tumor necrosis factor receptor superfamily , member Plasma Membrane transmembrane 19 receptor TP73 tumor protein p73 Nucleus transcription regulator US 9 ,944 ,986 B2 61 TABLE 1 - continued Rapamycin- sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type( s ) TPSD1 tryptase delta 1 Extracellular Space peptidase TRAM2 translocation associated membrane protein 2 unknown other TRIB1 tribbles homolog 1 ( Drosophila ) Cytoplasm kinase TRIM22 tripartite motif containing 22 Cytoplasm transcription regulator TRIM34 tripartite motif containing 34 Cytoplasm other TRIM49 tripartite motif containing 49 unknown other TRIM6 tripartite motif containing 6 Cytoplasm other TRIM72 tripartite motif containing 72 Cytoplasm other TRIML1 tripartite motif family - like 1 unknown other TRIML2 tripartite motif family - like 2 unknown other TRPM1 transient receptor potential cation channel , subfamily Plasma Membrane ion channel M , member 1 TRPM6 transient receptor potential cation channel , subfamily Plasma Membrane kinase M , member 6 TRPV1 transient receptor potential cation channel , subfamily Plasma Membrane ion channel V , member 1 TSPAN11 tetraspanin 11 unknown other TSPAN12 tetraspanin 12 Plasma Membrane transmembrane receptor TSPAN8 tetraspanin 8 Plasma Membrane other TTC18 tetratricopeptide repeat domain 18 unknown other TTC23L tetratricopeptide repeat domain 23 - like unknown other TTLL10 tubulin tyrosine ligase - like family , member 10 Extracellular Space other TTN (includes titin Cytoplasm kinase EG : 22138 ) TTPA tocopherol ( alpha ) transfer protein Cytoplasm transporter TTTY2 testis - specific transcript , Y - linked 2 ( non -protein Nucleus other coding ) TTTY8 testis - specific transcript, Y - linked 8 (non -protein unknown other coding ) TUSC5 tumor suppressor candidate 5 unknown other TXNRD2 thioredoxin reductase 2 Cytoplasm enzyme UACA uveal autoantigen with coiled -coil domains and Cytoplasm other ankyrin repeats UBE2M ubiquitin - conjugating enzyme E2M Cytoplasm enzyme UBE2R2 ubiquitin - conjugating enzyme E2R 2 unknown enzyme UBN2 ubinuclein 2 Nucleus other UCA1 urothelial cancer associated 1 (non -protein coding) unknown other UCP1 uncoupling protein 1 (mitochondrial , proton carrier ) Cytoplasm transporter UGT3A1 UDP glycosyltransferase 3 family, polypeptide A1 unknown enzyme ULK2 unc -51 - like kinase 2 ( C . elegans ) Cytoplasm kinase UNC80 unc - 80 homolog ( C . elegans ) unknown other USP11 ubiquitin specific peptidase 11 Nucleus peptidase USP38 ubiquitin specific peptidase 38 unknown peptidase UTS2 urotensin 2 Extracellular Space other UTS2D urotensin 2 domain containing Extracellular Space other VEZF1 vascular endothelial zinc finger 1 Nucleus transcription regulator VN1R4 vomeronasal 1 receptor 4 Plasma Membrane G -protein coupled receptor VPS54 ( includes vacuolar protein sorting 54 homolog ( S. cerevisiae ) unknown other EG : 245944 ) VSNL1 visinin - like 1 Cytoplasm other VSTM4 V - set and transmembrane domain containing 4 unknown other VWA1 von Willebrand factor A domain containing 1 Extracellular Space other VWAGB von Willebrand factor A domain containing 3B unknown other WASF2 WAS protein family , member 2 Cytoplasm other WDFY3 - AS2 WDFY3 antisense RNA 2 (non -protein coding ) unknown other WDR17 WD repeat domain 17 unknown other WDR45 WD repeat domain 45 unknown other WDR49 WD repeat domain 49 unknown other WDR65 WD repeat domain 65 unknown other WDR72 WD repeat domain 72 unknown other WDR96 WD repeat domain 96 unknown other WFDC11 WAP four- disulfide core domain 11 Extracellular Space other WFDC5 WAP four- disulfide core domain 5 Extracellular Space other WFDC6 WAP four- disulfide core domain 6 Extracellular Space other WFDC9 WAP four- disulfide core domain 9 Extracellular Space other WLS wntless homolog (Drosophila ) Cytoplasm other WNTSB wingless -type MMTV integration site family , member Extracellular Space other 8B WWP2 WW domain containing E3 ubiquitin protein ligase 2 Cytoplasm enzyme XIRP2 xin actin -binding repeat containing 2 unknown other XRN1 5 ' - 3 ' exoribonuclease 1 Cytoplasm enzyme US 9 ,944 ,986 B2 63 64 TABLE 1 - continued Rapamycin - sensitive genes ( 1051 genes ) Symbol Entrez Gene Name Location Type ( s ) XYLB xylulokinase homolog ( H . influenzae ) unknown kinase YPEL1 yippee - like 1 ( Drosophila ) Nucleus enzyme YPEL2 yippee - like 2 (Drosophila ) Nucleus other YPEL5 yippee - like 5 (Drosophila ) unknown other ZADH2 zinc binding alcohol dehydrogenase domain Cytoplasm enzyme containing 2 ZBTB10 zinc finger and BTB domain containing 10 Nucleus other ZBTB20 zinc finger and BTB domain containing 20 Nucleus other ZC3H6 zinc finger CCCH - type containing 6 unknown other ZCCHC12 zinc finger, CCHC domain containing 12 unknown other ZDHHC15 zinc finger , DHHC - type containing 15 unknown enzyme ZFYVE16 zinc finger , FYVE domain containing 16 Nucleus transporter ZIC4 Zic family member 4 Nucleus other ZMAT1 zinc finger , matrin - type 1 Nucleus other ZNF292 zinc finger protein 292 Nucleus transcription regulator ZNF385B zinc finger protein 385B Nucleus other ZNF445 zinc finger protein 445 Nucleus transcription regulator ZNF45 zinc finger protein 45 Nucleus transcription regulator ZNF471 zinc finger protein 471 Nucleus other ZNF572 zinc finger protein 572 Nucleus other ZNF695 zinc finger protein 695 Nucleus other ZNF704 zinc finger protein 704 unknown other ZNF711 zinc finger protein 711 Nucleus other ZNF804A zinc finger protein 804A unknown other ZNF81 zinc finger protein 81 Nucleus transcription regulator ZSCAN4 zinc finger and SCAN domain containing 4 Nucleus transcription regulator
TABLE 2 Rapamycin -sensitive genes differentially regulated in brain of Alzheimer 's disease patients with mild disease ( 112 known genes ) Symbol Entrez Gene Name Location Type ( s ) Fold Change ABTB1 ankyrin repeat and BTB (POZ ) domain containing 1 Cytoplasm translation regulator 1 . 057 ADAMTS2 ADAM metallopeptidase with thrombospondin type 1 motif , 2 Extracellular Space peptidase 1 . 083 ADAMTSL5 ADAMTS - like 5 Extracellular Space other 1 . 088 AHNAK2 AHNAK nucleoprotein 2 unknown other 1 . 056 B4GALT6 UDP -Gal : betaGlcNAc beta 1 , 4 - galactosyltransferase , Cytoplasm enzyme 1 .022 polypeptide 6 BARX2 BARX homeobox 2 Nucleus transcription regulator 1 . 077 BMP7 bone morphogenetic protein 7 Extracellular Space growth factor 1 . 035 BTNL9 butyrophilin - like 9 unknown other 1 . 056 C17orf99 chromosome 17 open reading frame 99 unknown other 1 . 032 Clorf127 chromosome 1 open reading frame 127 unknown other 1 . 074 CACNAID calcium channel, voltage- dependent , L type , alpha 1D Plasma Membrane ion channel 1 . 063 subunit CARD14 caspase recruitment domain family, member 14 Cytoplasm other 1 . 034 CC2D2A coiled -coil and C2 domain containing 2A unknown other 1 . 102 CCL1 chemokine ( C - C motif ) ligand 1 Extracellular Space cytokine 1 . 066 CCL11 chemokine ( C - C motif ) ligand 11 Extracellular Space cytokine 1 . 048 CCL2 chemokine ( C - C motif ) ligand 2 Extracellular Space cytokine 1. 061 CCR1 chemokine ( C — C motif) receptor 1 Plasma Membrane G -protein coupled 1 . 034 receptor CD69 CD69 molecule Plasma Membrane transmembrane 1 . 028 receptor CDKN2C cyclin - dependent kinase inhibitor 2C (p18 , inhibits CDK4 ) Nucleus transcription regulator 1 .056 CEACAM1 carcinoembryonic antigen - related cell adhesion molecule 1 Plasma Membrane transmembrane 1 . 079 ( includes others ) (biliary glycoprotein ) receptor CEP68 centrosomal protein 68 kDa Cytoplasm other 1 . 036 COL1A2 collagen , type I , alpha 2 Extracellular Space other 1 . 043 CPXM2 carboxypeptidase X (M14 family ) , member 2 Extracellular Space peptidase 1 .026 CREB3L4 CAMP responsive element binding protein 3 - like 4 Nucleus transcription regulator 1 . 064 CRP C - reactive protein , pentraxin -related Extracellular Space other 1 . 104 CTDSP1 CTD ( carboxy - terminal domain , RNA polymerase II , Nucleus phosphatase 1 . 098 polypeptide A ) small phosphatase 1 DCAF12L1 DDB1 and CUL4 associated factor 12 - like 1 unknown other 1 . 030 DCD dermcidin Extracellular Space other 1 . 159 DRP2 dystrophin related protein 2 Plasma Membrane other 1 . 043 US 9, 944 ,986 B2 65 66 TABLE 2 - continued Rapamycin - sensitive genes differentially regulated in brain of Alzheimer 's disease patients with mild disease ( 112 known genes ) Symbol Entrez Gene Name Location Type( s ) Fold Change EBF2 early B -cell factor 2 Nucleus other 1 . 028 ENTPD8 ectonucleoside triphosphate diphosphohydrolase 8 unknown enzyme 1 . 071 FAM43B family with sequence similarity 43 , member B unknown other 1 . 061 FAM64A family with sequence similarity 64 , member A Nucleus other 1. 214 FCGBP Fc fragment of IgG binding protein Extracellular Space other 1 . 057 FGF11 fibroblast growth factor 11 Extracellular Space growth factor 1 . 169 FJX1 four jointed box 1 ( Drosophila ) Extracellular Space other 1 . 123 FLJ35946 uncharacterized protein FLJ35946 unknown other 1. 148 FN3K fructosamine 3 kinase Cytoplasm kinase 1 . 258 GLI2 GLI family zinc finger 2 Nucleus transcription regulator 1 .032 GLP1R glucagon - like peptide 1 receptor Plasma Membrane G -protein coupled 1 . 031 receptor GLRA1 glycine receptor , alpha 1 Plasma Membrane ion channel 1 . 041 GPR176 G protein - coupled receptor 176 Plasma Membrane G - protein coupled 1 . 092 receptor HCAR3 hydroxycarboxylic acid receptor 3 Plasma Membrane G -protein coupled 1 . 041 receptor HDAC5 histone deacetylase 5 Nucleus transcription regulator 1 . 112 HHIPL1 HHIP - like 1 unknown other 1 . 057 HMGCS2 3 -hydroxy - 3 -methylglutaryl - CoA synthase 2 (mitochondrial ) Cytoplasm enzyme 1 . 044 HOXD10 homeobox D10 Nucleus transcription regulator 1 .024 HYDIN HYDIN , axonemal central pair apparatus protein unknown other 1 . 077 IL6 interleukin 6 ( interferon , beta 2 ) Extracellular Space cytokine 1 . 034 KLF2 Kruppel- like factor 2 ( lung ) Nucleus transcription regulator 1 . 084 LECT1 leukocyte cell derived chemotaxin 1 Extracellular Space other 1 . 062 LINC00473 long intergenic non -protein coding RNA 473 unknown other 1 . 097 LOC100129775 uncharacterized LOC100129775 unknown other 1 . 047 LOC100505890 uncharacterized LOC100505890 unknown other 1 . 025 LOC100506206 uncharacterized LOC100506206 unknown other 1 . 192 LOC100506236 uncharacterized LOC100506236 unknown other 1 . 126 LOC100507492 uncharacterized LOC100507492 unknown other 1 . 086 LOC100507520 uncharacterized LOC100507520 unknown other 1 . 495 LOC285740 uncharacterized LOC285740 unknown other 1 . 027 LOC389043 uncharacterized LOC389043 unknown other 1 . 330 LOC400752 uncharacterized LOC400752 unknown other 1 . 115 LOC401317 uncharacterized LOC401317 unknown other 1 . 034 LOC728724 hCG1814486 unknown other 1 . 051 LOXL4 lysyl oxidase - like 4 Extracellular Space enzyme 1 . 122 LUM lumican Extracellular Space other 1 . 073 LYPD2 LY6 / PLAUR domain containing 2 unknown other 1 . 101 MAPK4 mitogen -activated protein kinase 4 Cytoplasm kinase 1 . 103 MFAP5 microfibrillar associated protein 5 Extracellular Space other 1 . 024 MGC24103 uncharacterized MGC24103 unknown other 1 . 045 MGC39545 uncharacterized LOC403312 unknown other 1 . 026 MOXD1 monooxygenase , DBH - like 1 Cytoplasm enzyme 1 . 124 MYF6 myogenic factor 6 ( herculin ) Nucleus transcription regulator 1 . 127 NCKAP1 NCK -associated protein 1 Plasma Membrane other 1 . 043 NR3C1 nuclear receptor subfamily 3 , group C , member 1 Nucleus ligand -dependent 1 . 058 ( glucocorticoid receptor ) nuclear receptor NTSR1 neurotensin receptor 1 (high affinity ) Plasma Membrane G -protein coupled 1 . 058 receptor OR52B2 olfactory receptor , family 52 , subfamily B , member 2 Plasma Membrane G - protein coupled 1 . 051 receptor OTOGL otogelin - like unknown other 1 . 039 PAOX polyamine oxidase ( exo -N4 - amino ) Cytoplasm enzyme 1 . 043 PBX1 pre - B - cell leukemia homeobox 1 Nucleus transcription regulator 1 .021 PHLDA3 pleckstrin homology - like domain , family A , member 3 Plasma Membrane other 1 . 056 PIM1 pim - 1 oncogene Cytoplasm kinase 1 . 041 PLD4 phospholipase D family , member 4 unknown enzyme 1 . 087 POU4F2 POU class 4 homeobox 2 Nucleus transcription regulator 1 . 040 PRKCH protein kinase C , eta Cytoplasm kinase 1 . 169 PRODH2 proline dehydrogenase (oxidase ) 2 Cytoplasm enzyme 1 . 056 PVRL3 poliovirus receptor -related 3 Plasma Membrane other 1 . 064 RELN reelin Extracellular Space peptidase 1 . 113 RHO rhodopsin Plasma Membrane G - protein coupled 1 . 024 receptor RHOT1 ras homolog family member T1 Cytoplasm enzyme 1 . 029 RPSÓKB2 ribosomal protein S6 kinase , 70 kDa, polypeptide 2 Cytoplasm kinase 1 . 077 RUNX1T1 runt- related transcription factor 1 ; translocated to , 1 ( cyclin Nucleus transcription regulator 1 . 053 D - related ) SEMA4C sema domain , immunoglobulin domain (Ig ) , transmembrane Plasma Membrane other 1 . 135 domain ( TM ) and short cytoplasmic domain , ( semaphorin ) 4C SEMASA sema domain , seven thrombospondin repeats ( type 1 and Plasma Membrane transmembrane 1 . 092 type 1 - like ) , transmembrane domain ( TM ) and short receptor cytoplasmic domain , (semaphorin ) 5A US 9, 944 ,986 B2 68 TABLE 2 -continued Rapamycin - sensitive genes differentially regulated in brain of Alzheimer 's disease patients with mild disease ( 112 known genes ) Symbol Entrez Gene Name Location Type( s ) Fold Change SERPIND1 serpin peptidase inhibitor, clade D (heparin cofactor) , Extracellular Space other 1 . 030 member 1 SERPINE1 serpin peptidase inhibitor , clade E ( nexin , plasminogen Extracellular Space other 1 . 091 activator inhibitor type 1 ) , member 1 SHE Src homology 2 domain containing E Cytoplasm other 1 . 030 SLC18A3 solute carrier family 18 (vesicular acetylcholine) , member 3 Plasma Membrane transporter 1 . 056 SLC22A18 solute carrier family 22 , member 18 Plasma Membrane transporter 1 . 173 SLC22A7 solute carrier family 22 (organic anion transporter) , member 7 Plasma Membrane transporter 1 . 077 SLC38A3 solute carrier family 38 , member 3 Plasma Membrane transporter 1 . 033 SMAD3 SMAD family member 3 Nucleus transcription regulator 1 . 042 STARD13 STAR -related lipid transfer ( START) domain containing 13 Cytoplasm other 1. 269 TM4SF4 transmembrane 4 L six family member 4 Plasma Membrane other 1 . 024 TNFRSF100 tumor necrosis factor receptor superfamily , member 10c , Plasma Membrane transmembrane 1 .030 decoy without an intracellular domain receptor TRIB1 tribbles homolog 1 (Drosophila ) Cytoplasm kinase 1 . 041 TRPV1 transient receptor potential cation channel , subfamily V , Plasma Membrane ion channel 1 .037 member 1 TTC18 tetratricopeptide repeat domain 18 unknown other 1 . 072 UCA1 urothelial cancer associated 1 (non - protein coding ) unknown other 1 . 041 WFDC9 WAP four- disulfide core domain 9 Extracellular Space other 1 . 052 XRN1 5 '- 3 ' exoribonuclease 1 Cytoplasm enzyme 1 . 027 ZADH2 zinc binding alcohol dehydrogenase domain containing 2 Cytoplasm enzyme 1 . 129 ZBTB20 zinc finger and BTB domain containing 20 Nucleus other 1 .044
TABLE 3 Rapamycin - sensitive genes differentially regulated in brain of Alzheimer' s disease patients with advanced disease (178 known genes ) Symbol Entrez Gene Name Location Type( s ) Fold Change ABLIM2 actin binding LIM protein family , member 2 Cytoplasm other 0 . 905 ABTB1 ankyrin repeat and BTB ( POZ ) domain containing 1 Cytoplasm translation regulator 1 . 058 ACSL6 acyl- CoA synthetase long -chain family member 6 Cytoplasm enzyme 0 . 937 ACTRT1 actin -related protein T1 Cytoplasm other 1 . 025 ?CVR2B activin A receptor, type IIB Plasma Membrane kinase 1 . 038 ADAMTS2 ADAM metallopeptidase with thrombospondin type 1 Extracellular Space peptidase 1 . 082 motif , 2 ADAMTSL5 ADAMTS - like 5 Extracellular Space other 1 . 094 AHNAK2 AHNAK nucleoprotein 2 unknown other 1 . 056 ANKRD36BP2 ankyrin repeat domain 36B pseudogene 2 unknown other 0 . 913 AP1S1 adaptor- related protein complex 1 , sigma 1 subunit Cytoplasm transporter 0 . 955 ATAD3A ATAD3B ATPase family , AAA domain containing 3A Nucleus other 1 . 078 ATF7IP activating transcription factor 7 interacting protein Nucleus transcription regulator 0 . 934 ATRNL1 attractin - like 1 unknown other 0 . 865 BARX2 BARX homeobox 2 Nucleus transcription regulator 1 . 080 BCOR BCL6 corepressor Nucleus transcription regulator 1 . 032 BMP7 bone morphogenetic protein 7 Extracellular Space growth factor 1 . 046 BPI bactericidal/ permeability -increasing protein Plasma Membrane transporter 1 . 036 BTNL9 butyrophilin - like 9 unknown other 1 . 063 C17orf99 chromosome 17 open reading frame 99 unknown other 1 .039 C18orf26 chromosome 18 open reading frame 26 unknown other 0 . 960 Clorf127 chromosome 1 open reading frame 127 unknown other 1 . 087 C3orf80 chromosome 3 open reading frame 80 unknown other 0 . 952 CACNAID calcium channel, voltage- dependent , L type, alpha 1D Plasma Membrane ion channel 1 . 070 subunit CARD14 caspase recruitment domain family , member 14 Cytoplasm other 1 .025 CC2D2A coiled - coil and C2 domain containing 2A unknown other 1 . 098 CCL1 chemokine ( C — C motif ) ligand 1 Extracellular Space cytokine 1 . 063 CCL11 chemokine ( C - C motif ) ligand 11 Extracellular Space cytokine 1. 039 CCL2 chemokine ( C - C motif ) ligand 2 Extracellular Space cytokine 1 . 082 CCNB2 cyclin B2 Cytoplasm other 1 . 073 CCR1 chemokine ( C — C motif) receptor 1 Plasma Membrane G - protein coupled 1 .032 receptor CEACAM1 carcinoembryonic antigen -related cell adhesion molecule Plasma Membrane transmembrane 1 . 081 ( includes others ) 1 (biliary glycoprotein ) receptor CEP68 centrosomal protein 68 kDa Cytoplasm other 1 . 040 CLU clusterin Extracellular Space other 0 . 911 CPLX2 complexin 2 Cytoplasm other 0 . 918 CPXM2 carboxypeptidase X (M14 family ), member 2 Extracellular Space peptidase 1 . 030 US 9, 944 ,986 B2 69 70 TABLE 3 - continued Rapamycin - sensitive genes differentially regulated in brain of Alzheimer ' s disease patients with advanced disease ( 178 known genes ) Symbol Entrez Gene Name Location Type( s ) Fold Change CREB3L4 CAMP responsive element binding protein 3 - like 4 Nucleus transcription regulator 1 . 054 CRP C - reactive protein , pentraxin - related Extracellular Space other 1 . 111 CTDSP1 CTD (carboxy - terminal domain , RNA polymerase II , Nucleus phosphatase 1 . 119 polypeptide A ) small phosphatase 1 CUX1 cut- like homeobox 1 Nucleus transcription regulator 0 . 969 DCAF12L1 DDB1 and CUL4 associated factor 12 - like 1 unknown other 1 .038 DCD dermcidin Extracellular Space other 1 . 152 DCLK1 doublecortin - like kinase 1 Plasma Membrane kinase 0 . 939 DLG2 discs, large homolog 2 (Drosophila ) Plasma Membrane kinase 0 . 917 DNAH6 dynein , axonemal, heavy chain 6 unknown other 0 . 957 DRP2 dystrophin related protein 2 Plasma Membrane other 1 . 038 DYNCZLI1 dynein , cytoplasmic 2 , light intermediate chain 1 Cytoplasm other 0 . 946 DZIP3 DAZ interacting protein 3 , zinc finger Cytoplasm enzyme 0 . 905 EBF2 early B - cell factor 2 Nucleus other 1 . 024 EIF4E eukaryotic translation initiation factor 4E Cytoplasm translation regulator 0 . 920 ELAVL4 ELAV ( embryonic lethal, abnormal vision , Drosophila ) - like Cytoplasm other 0 . 931 4 (Hu antigen D ) ENTPD8 ectonucleoside triphosphate diphosphohydrolase 8 unknown enzyme 1 . 063 FAM155A family with sequence similarity 155 , member A unknown other 0 . 860 FAM171B family with sequence similarity 171 , member B unknown other 0 . 948 FAM64A family with sequence similarity 64 , member A Nucleus other 1 . 154 FAT3 FAT tumor suppressor homolog 3 (Drosophila ) unknown other 0 . 954 FGF11 fibroblast growth factor 11 Extracellular Space growth factor 1 . 185 FJX1 four jointed box 1 ( Drosophila ) Extracellular Space other 1 . 104 FLJ35946 uncharacterized protein FLJ35946 unknown other 1 . 137 FN3K fructosamine 3 kinase Cytoplasm kinase 1 . 269 GABBR2 gamma- aminobutyric acid (GABA ) B receptor, 2 Plasma Membrane G - protein coupled 0 . 862 receptor GALNT10 UDP- N - acetyl- alpha- D -galactosamine : polypeptide N Cytoplasm enzyme 1 . 064 acetylgalactosaminyltransferase 10 (GalNAc - T10 ) GFAP glial fibrillary acidic protein Cytoplasm other 1 . 091 GIGYF1 GRB10 interacting GYF protein 1 unknown other 1 . 042 GLI2 GLI family zinc finger 2 Nucleus transcription regulator 1 . 033 GLRA1 glycine receptor, alpha 1 Plasma Membrane ion channel 1 . 043 GNGS guanine nucleotide binding protein (G protein ) , gamma 8 Plasma Membrane enzyme 1 . 059 GPM6A glycoprotein M6A Plasma Membrane ion channel 0 . 920 GPR176 G protein - coupled receptor 176 Plasma Membrane G -protein coupled 1 . 098 receptor GSGIL GSG1- like unknown other 1 . 025 HCAR3 hydroxycarboxylic acid receptor 3 Plasma Membrane G - protein coupled 1 . 053 receptor HDAC5 histone deacetylase 5 Nucleus transcription regulator 1. 125 HHIPL1 HHIP - like 1 unknown other 1 . 063 HIST1H4A histone cluster 1 , H4a Nucleus other 0 . 894 ( includes others ) HMGCS2 3 -hydroxy -3 - methylglutaryl - CoA synthase 2 Cytoplasm enzyme 1 . 034 (mitochondrial ) HOXD10 homeobox D10 Nucleus transcription regulator 1 .038 HYDIN HYDIN , axonemal central pair apparatus protein unknown other 1 . 090 ILIRAPL1 interleukin 1 receptor accessory protein - like 1 Plasma Membrane transmembrane 1 . 029 receptor IL6 interleukin 6 ( interferon , beta 2 ) Extracellular Space cytokine 1 .037 aIRS2 insulin receptor substrate 2 Cytoplasm enzyme 0 . 925 IRS2 insulin receptor substrate 2 Cytoplasm enzyme 0 . 941 IRX4 iroquois homeobox 4 Nucleus transcription regulator 1 . 032 ITGBL1 integrin , beta - like 1 ( with EGF - like repeat domains ) unknown other 1 . 038 KCNAB1 potassium voltage - gated channel, shaker- related Plasma Membrane ion channel 0 . 936 t subfamily , beta member 1 KCND2 potassium voltage- gated channel , Shal- related subfamily , Plasma Membrane ion channel 0 . 958 member 2 KCNG4 potassium voltage- gated channel, subfamily G , member 4 Plasma Membrane ion channel 1 . 152 KIAA1683 KIAA1683 Cytoplasm other 1 . 046 KLF2 Kruppel- like factor 2 ( lung ) Nucleus transcription regulator 1 . 103 KRTAP9 - 2 keratin associated protein 9 - 2 unknown other 1 . 022 LECT1 leukocyte cell derived chemotaxin 1 Extracellular Space other 1. 060 LINC00473 long intergenic non -protein coding RNA 473 unknown other 1 . 085 LOC100129775 uncharacterized LOC100129775 unknown other 1 . 051 LOC100287803 uncharacterized LOC100287803 unknown other 0 . 854 LOC100506206 uncharacterized LOC100506206 unknown other 1 . 199 LOC100506236 uncharacterized LOC100506236 unknown other 1 . 127 LOC100507492 uncharacterized LOC100507492 unknown other 1 . 080 LOC100507520 uncharacterized LOC100507520 unknown other 1 . 497 LOC285740 uncharacterized LOC285740 unknown other 1 . 024 LOC389043 uncharacterized LOC389043 unknown other 1 . 255 LOC400752 uncharacterized LOC400752 unknown other 1 . 113 LOC401317 uncharacterized LOC401317 unknown other 1 . 037 US 9, 944 ,986 B2
TABLE 3 - continued Rapamycin - sensitive genes differentially regulated in brain of Alzheimer ' s disease patients with advanced disease ( 178 known genes ) Symbol Entrez Gene Name Location Type( s ) Fold Change LOC728323 uncharacterized LOC728323 unknown other 0 . 921 LOC728724 hCG1814486 unknown other 1 . 070 LOXL4 lysyl oxidase - like 4 Extracellular Space enzyme 1 . 136 LRRC71 leucine rich repeat containing 71 unknown other 1 . 052 LUM lumican Extracellular Space other 1 . 069 LYPD2 LY6 /PLAUR domain containing 2 unknown other 1 . 102 MAPK1 mitogen -activated protein kinase 1 Cytoplasm kinase 0 . 912 MAPK4 mitogen - activated protein kinase 4 Cytoplasm kinase 1 . 100 MBD3L2 ( includes methyl- CpG binding domain protein 3 - like 2 unknown other 1 .025 others ) ????1 multiple C2 domains , transmembrane 1 unknown other 0 . 914 MECOM MDS1 and EV11 complex locus Nucleus transcription regulator 1 . 024 MFAP5 microfibrillar associated protein 5 Extracellular Space other 1 . 026 MGC24103 uncharacterized MGC24103 unknown other 1 . 050 MMP16 matrix metallopeptidase 16 (membrane - inserted ) Extracellular Space peptidase 0 . 928 MOXD1 monooxygenase , DBH - like 1 Cytoplasm enzyme 1 . 103 MYF6 myogenic factor 6 (herculin ) Nucleus transcription regulator 1 . 136 NAV2 neuron navigator 2 Nucleus other 1 . 037 NCKAP1 NCK -associated protein 1 Plasma Membrane other 1 . 043 NHSL1 NHS - like 1 unknown other 0 . 960 NR3C1 nuclear receptor subfamily 3, group C , member 1 Nucleus ligand -dependent 1 . 078 ( glucocorticoid receptor ) nuclear receptor NTSR1 neurotensin receptor 1 (high affinity ) Plasma Membrane G - protein coupled 1 . 053 receptor OIP5 - AS1 OIP5 antisense RNA 1 (non -protein coding ) unknown other 0 . 888 OR52B2 olfactory receptor, family 52 , subfamily B , member 2 Plasma Membrane G - protein coupled 1 . 065 receptor OTOGL otogelin - like unknown other 1 . 031 PEX5L peroxisomal biogenesis factor 5 - like Cytoplasm ion channel 0 . 932 PHLDA3 pleckstrin homology - like domain , family A , member 3 Plasma Membrane other 1 . 067 PIM1 pim - 1 oncogene Cytoplasm kinase 1 . 054 PLD4 phospholipase D family , member 4 unknown enzyme 1 . 085 POU4F2 POU class 4 homeobox 2 Nucleus transcription regulator 1 . 034 PRKCH protein kinase C , eta Cytoplasm kinase 1 . 156 PRODH2 proline dehydrogenase (oxidase ) 2 Cytoplasm enzyme 1 . 062 PVRL3 poliovirus receptor- related 3 Plasma Membrane other 1 . 066 PYCR1 pyrroline - 5 - carboxylate reductase 1 Cytoplasm enzyme 1 . 048 RAB3C (includes RAB3C , member RAS oncogene family Cytoplasm enzyme 0 . 941 EG : 115827 ) RELN reelin Extracellular Space peptidase 1 . 081 RMI2 RMI2 , RecQ mediated genome instability 2 , homolog ( S . unknown other 1 . 069 cerevisiae ) RND3 Rho family GTPase 3 Cytoplasm enzyme 1 . 026 RNF128 ring finger protein 128 , E3 ubiquitin protein ligase Cytoplasm enzyme 0 . 952 RPSÁKB2 ribosomal protein S6 kinase , 70 kDa , polypeptide 2 Cytoplasm kinase 1 . 098 RUNX1T1 runt - related transcription factor 1 ; translocated to , 1 ( cyclin Nucleus transcription regulator 1 . 055 D - related ) SCGN secretagogin , EF - hand calcium binding protein Cytoplasm other 1 . 037 SCNSA sodium channel , voltage gated , type VIII, alpha subunit Plasma Membrane ion channel 0 . 924 SEMA4C sema domain , immunoglobulin domain ( Ig ) , Plasma Membrane other 1 . 146 transmembrane domain ( TM ) and short cytoplasmic domain , (semaphorin ) 4C SEMASA sema domain , seven thrombospondin repeats ( type 1 and Plasma Membrane transmembrane 1 . 083 type 1 - like ) , transmembrane domain ( TM ) and short receptor cytoplasmic domain , (semaphorin ) 5A SERPINA10 serpin peptidase inhibitor, clade A ( alpha - 1 antiproteinase , Extracellular Space other 1 . 029 antitrypsin ), member 10 SERPINDI serpin peptidase inhibitor , clade D (heparin cofactor ), Extracellular Space other 1 . 036 member 1 SERPINE1 serpin peptidase inhibitor, clade E (nexin , plasminogen Extracellular Space other 1 . 093 activator inhibitor type 1 ) , member 1 SHE Src homology 2 domain containing E Cytoplasm other 1 .043 SLC18A3 solute carrier family 18 ( vesicular acetylcholine ) , member 3 Plasma Membrane transporter 1 . 056 SLC22A18 solute carrier family 22 , member 18 Plasma Membrane transporter 1 . 176 SLC22A7 solute carrier family 22 ( organic anion transporter ), Plasma Membrane transporter 1 . 082 member 7 SLC25A27 solute carrier family 25 , member 27 Cytoplasm transporter 0 . 952 SLC38A3 solute carrier family 38 , member 3 Plasma Membrane transporter 1 . 046 SLITRK1 SLIT and NTRK - like family, member 1 unknown other 0 . 917 SLITRK1 SLIT and NTRK - like family , member 1 unknown other 0 . 948 SMAD3 SMAD family member 3 Nucleus transcription regulator 1 . 042 SORBS2 sorbin and SH3 domain containing 2 Plasma Membrane other 0 . 961 STAG3L1 stromal antigen 3 - like 1 unknown other 1 . 088 STARD13 STAR - related lipid transfer (START ) domain containing 13 Cytoplasm other 1 . 221 STX1B syntaxin 1B Plasma Membrane ion channel 0 . 915 SYCP3 synaptonemal complex protein 3 Nucleus other 1 . 023 US 9, 944 ,986 B2 73 74 TABLE 3 - continued Rapamycin - sensitive genes differentially regulated in brain of Alzheimer ' s disease patients with advanced disease ( 178 known genes ) Symbol Entrez Gene Name Location Type( s ) Fold Change TAOK1 TAO kinase 1 Cytoplasm kinase 0 . 965 TGM4 transglutaminase 4 (prostate ) Extracellular Space enzyme 1 . 086 TIMM17B translocase of inner mitochondrial membrane 17 homolog Cytoplasm transporter 1 . 035 B ( yeast ) TMEM56 transmembrane protein 56 unknown other 0 . 947 TNFRSF100 tumor necrosis factor receptor superfamily , member 10c , Plasma Membrane transmembrane 1 . 028 decoy without an intracellular domain receptor TP73 tumor protein p73 Nucleus transcription regulator 1 . 045 TRPV1 transient receptor potential cation channel , subfamily V , Plasma Membrane ion channel 1 .032 member 1 TSC2 tuberous sclerosis 2 Cytoplasm other 1 . 045 UCA1 urothelial cancer associated 1 (non - protein coding ) unknown other 1 . 040 VEGFA vascular endothelial growth factor A Extracellular Space growth factor 1 . 090 VSNL1 visinin - like 1 Cytoplasm other 0 . 848 WFDC11 WAP four- disulfide core domain 11 Extracellular Space other 1 .030 XRN1 5 '- 3 ' exoribonuclease 1 Cytoplasm enzyme 1 . 023 YPEL1 yippee- like 1 ( Drosophila ) Nucleus enzyme 0 . 934 ZADH2 zinc binding alcohol dehydrogenase domain containing 2 Cytoplasm enzyme 1 . 133 ZBTB20 zinc finger and BTB domain containing 20 Nucleus other 1 . 052 ZC3H6 zinc finger CCCH - type containing 6 unknown other 0 . 928
TABLE 4 Rapamycin - sensitive genes differentially regulated in brain of Alzheimer ' s disease patients with mild disease - new targets Symbol Entrez Gene Name Location Type( s ) Fold Change ABTB1 ankyrin repeat and BTB ( POZ ) domain containing 1 Cytoplasm translation regulator 1 .057 AHNAK2 AHNAK nucleoprotein 2 unknown other 1 . 056 BARX2 BARX homeobox 2 Nucleus transcription regulator 1 . 077 BTNL9 butyrophilin - like 9 unknown other 1 . 056 CACNA1D calcium channel, voltage- dependent , L type , alpha 1D Plasma Membrane ion channel 1 . 063 subunit CARD14 caspase recruitment domain family , member 14 Cytoplasm other 1 .034 CC2D2A coiled - coil and C2 domain containing 2A unknown other 1 . 102 CEP68 centrosomal protein 68 kDa Cytoplasm other 1 . 036 CPXM2 carboxypeptidase X (M14 family ) , member 2 Extracellular Space peptidase 1 .026 CREB3L4 CAMP responsive element binding protein 3 - like 4 Nucleus transcription regulator 1 . 064 CTDSP1 CTD ( carboxy - terminal domain , RNA polymerase II, Nucleus phosphatase 1 . 098 polypeptide A ) small phosphatase 1 DCAF12L1 DDB1 and CUL4 associated factor 12 - like 1 unknown other 1 .030 FAM64A family with sequence similarity 64 , member A Nucleus other 1 . 214 FCGBP Fc fragment of IgG binding protein Extracellular Space other 1 . 057 FJX1 four jointed box 1 ( Drosophila ) Extracellular Space other 1 . 123 FN3K fructosamine 3 kinase Cytoplasm kinase 1. 258 GLI2 GLI family zinc finger 2 Nucleus transcription regulator 1 .032 GLRA1 glycine receptor , alpha 1 Plasma Membrane ion channel 1 . 041 GPR176 G protein - coupled receptor 176 Plasma Membrane G -protein coupled 1 . 092 receptor HHIPL1 HHIP - like 1 unknown other 1 . 057 HOXD10 homeobox D10 Nucleus transcription regulator 1 .024 HYDIN HYDIN , axonemal central pair apparatus protein unknown other 1 . 077 KLF2 Kruppel - like factor 2 ( lung ) Nucleus transcription regulator 1 . 084 MFAP5 microfibrillar associated protein 5 Extracellular Space other 1 . 024 PHLDA3 pleckstrin homology - like domain , family A , member 3 Plasma Membrane other 1 .056 POU4F2 POU class 4 homeobox 2 Nucleus transcription regulator 1 .040 RHO rhodopsin Plasma Membrane G - protein coupled 1 . 024 receptor RUNX1T1 runt- related transcription factor 1; translocated to , 1 (cyclin Nucleus transcription regulator 1 . 053 D - related ) STARD13 StAR -related lipid transfer (START ) domain containing 13 Cytoplasm other 1 . 269 TRIB1 tribbles homolog 1 ( Drosophila ) Cytoplasm kinase 1 . 041
TABLE 5 Rapamycin - sensitive genes differentially regulated in brain of Alzheimer ' s disease patients with advanced disease - new targets Symbol Entrez Gene Name Location TypeTuners( s ) Fold Change ABLIM2 actin binding LIM protein family , member 2 Cytoplasm other 0 . 905 ABTB1 ankyrin repeat and BTB (POZ ) domain containing 1 Cytoplasm translation regulator 1 . 058 US 9, 944 ,986 B2 75 76 TABLE 5 - continued Rapamycin -sensitive genes differentially regulated in brain of Alzheimer 's disease patients with advanced disease - new targets Symbol Entrez Gene Name Location Type( s ) Fold Change ACSL6 acyl- CoA synthetase long - chain family member 6 Cytoplasm enzyme 0 . 937 ACTRT1 actin - related protein T1 Cytoplasm other 1 . 025 AHNAK2 AHNAK nucleoprotein 2 unknown other 1 . 056 AP1S1 adaptor- related protein complex 1 , sigma 1 subunit Cytoplasm transporter 0 . 955 ATAD3A / ATAD3B ATPase family , AAA domain containing 3A Nucleus other 1 . 078 ATFTIP activating transcription factor 7 interacting protein Nucleus transcription regulator 0 . 934 ATRNL1 attractin - like 1 unknown other 0 . 865 BARX2 BARX homeobox 2 Nucleus transcription regulator 1 . 080 BCOR BCL6 corepressor Nucleus transcription regulator 1 . 032 BTNL9 butyrophilin - like 9 unknown other 1 .063 CACNAID calcium channel , voltage -dependent , L type, alpha 1D Plasma Membrane ion channel 1 . 070 subunit CARD14 caspase recruitment domain family , member 14 Cytoplasm other 1 . 025 CC2D2A coiled - coil and C2 domain containing 2A unknown other 1 . 098 CEP68 centrosomal protein 68 kDa Cytoplasm other 1 .040 CPXM2 carboxypeptidase X (M14 family ) , member 2 Extracellular Space peptidase 1 . 030 CREB3L4 CAMP responsive element binding protein 3 - like 4 Nucleus transcription regulator 1 . 054 CTDSP1 CTD ( carboxy - terminal domain , RNA polymerase II , Nucleus phosphatase 1 . 119 polypeptide A ) small phosphatase 1 DCLK1 doublecortin - like kinase 1 Plasma Membrane kinase 0 . 939 DLG2 discs, large homolog 2 (Drosophila ) Plasma Membrane kinase 0 .917 DNAH6 dynein , axonemal, heavy chain 6 unknown other 0 . 957 DYNCZLI1 dynein , cytoplasmic 2 , light intermediate chain 1 Cytoplasm other 0 . 946 DZIP3 DAZ interacting protein 3 , zinc finger Cytoplasm enzyme 0 . 905 ELAVL4 ELAV ( embryonic lethal , abnormal vision , Drosophila )- like Cytoplasm other 0 . 931 4 (Hu antigen D ) FAM155A family with sequence similarity 155 , member A unknown other 0 . 860 FAM64A family with sequence similarity 64 , member A Nucleus other 1 . 154 FAT3 FAT tumor suppressor homolog 3 (Drosophila ) unknown other 0 . 954 FJX1 four jointed box 1 (Drosophila ) Extracellular Space other 1 . 104 FN3K fructosamine 3 kinase Cytoplasm kinase 1 . 269 GABBR2 gamma- aminobutyric acid (GABA ) B receptor, 2 Plasma Membrane G - protein coupled 0 . 862 receptor GALNT10 UDP - N - acetyl- alpha - D -galactosamine : polypeptide N Cytoplasm enzyme 1 . 064 acetylgalactosaminyltransferase 10 (GalNAc - T10 ) GLI2 GLI family zinc finger 2 Nucleus transcription regulator 1 . 033 GLRA1 glycine receptor, alpha 1 Plasma Membrane ion channel 1 . 043 GPR176 G protein -coupled receptor 176 Plasma Membrane G - protein coupled 1 . 098 receptor GSGIL GSG1- like unknown other 1 . 025 HHIPL1 HHIP - like 1 unknown other 1 . 063 HOXD10 homeobox D10 Nucleus transcription regulator 1 .038 HYDIN HYDIN , axonemal central pair apparatus protein unknown other 1 .090 IRX4 iroquois homeobox 4 Nucleus transcription regulator 1 . 032 KLF2 Kruppel- like factor 2 ( lung) Nucleus transcription regulator 1 . 103 KRTAP9 - 2 keratin associated protein 9 - 2 unknown other 1 . 022 MBD3L2 ( includes methyl- CpG binding domain protein 3 - like 2 unknown other 1 . 025 others ) ????1 multiple C2 domains, transmembrane 1 unknown other 0 . 914 MFAP5 microfibrillar associated protein 5 Extracellular Space other 1 . 026 MMP16 matrix metallopeptidase 16 (membrane - inserted ) Extracellular Space peptidase 0 . 928 PEX5L peroxisomal biogenesis factor 5 - like Cytoplasm ion channel 0 . 932 PHLDA3 pleckstrin homology - like domain , family A , member 3 Plasma Membrane other 1 . 067 POU4F2 POU class 4 homeobox 2 Nucleus transcription regulator 1 . 034 PYCR1 pyrroline - 5 - carboxylate reductase 1 Cytoplasm enzyme 1 . 048 RND3 Rho family GTPase 3 Cytoplasm enzyme 1 . 026 RNF128 ring finger protein 128 , E3 ubiquitin protein ligase Cytoplasm enzyme 0 . 952 RUNX1T1 runt- related transcription factor 1 ; translocated to , 1 (cyclin Nucleus transcription regulator 1 . 055 D -related ) SLITRK1 SLIT and NTRK - like family , member 1 unknown other 0 . 917 SLITRK1 SLIT and NTRK - like family , member 1 unknown other 0 . 948 SORBS2 sorbin and SH3 domain containing 2 Plasma Membrane other 0 . 961 STAG3L1 stromal antigen 3 - like 1 unknown other 1 . 088 STARD13 STAR - related lipid transfer (START ) domain containing 13 Cytoplasm other 1 . 221 STX1B syntaxin 1B Plasma Membrane ion channel 0 . 915 TGM4 transglutaminase 4 (prostate ) Extracellular Space enzyme 1 . 086 TMEM56 transmembrane protein 56 unknown other 0 . 947 WFDC11 WAP four- disulfide core domain 11 Extracellular Space other 1 . 030 YPEL1 yippee -like 1 (Drosophila ) Nucleus enzyme 0 . 934 US 9 ,944 ,986 B2 77 78 The invention will now be further understood with refer at 4° C . to produce the DNA pellet. The pink supernatant ence to the following non - limiting examples . was transferred to a clean eppendorf in preparation for protein extraction . The DNA pellet was washed three times , EXAMPLES 1 hour per wash , in 0 . 1M sodium citrate in 10 % ethanol, 5 with centrifugation at 12, 000 g for 5 minutes between Example 1 washes . Following a 30 minute wash in 75 % ethanol, the pellet was centrifuged at 2 ,000 g for 5 minutes to allow wash Gene Expression Microarray Analysis of Brain removal , and the pellet air dried overnight . The pellet was Samples from Alzheimer ' S Disease Patients dissolved in nuclease free water for 24 hours, centrifuged at 10 1 . 1 Patients and Biomaterials 16 ,000 g for 10 minutes, and supernatant transferred to a Human brain tissue collected by the Oxford Project to clean tube to separate DNA from any insoluble material. The Investigate Memory and Aging (OPTIMA ) was made avail DNA was stored at 4° C . able through the Thomas Willis brain bank . In total, samples The protein was precipitated from the pink layer by 15 from 252 brains were available from a mix of elderly 15 minute incubation with 3 volumes acetone prior to centrifu controls and patients suffer from preclinical, mild and severe gation at 12 ,000 g for 10 min at 4° C . The protein pellet was AD at the time of death (as determined by Braak staging ) . subjected to three 10 minute washes in 0 .3M guanidine The number of cases included in the study was based on hydrochloride in 95 % ethanol and 2 . 5 % glycerol, and dis availability, not statistical power calculations, as the out- solved in radio - immunoprecipitation ( RIPA ) buffer (compo come, and hence number of patients in each group -of - 20 sition : 0 . 1 M sodium chloride, 0 .01 M Tris hydrochloride , interest, was unknown . The tissue was snap frozen at the 1 : 500 EDTA , 400 ug /ml phenylmethanesulfonylfluoride , 2 time of autopsy : and available from the lateral temporal lobe ug /ml aprotinin and 1 % sodium dodecyl sulphate ) . The ( severely affected by AD pathology ), frontal lobe (affected protein was stored at - 20° C . by AD pathology ) and occipital lobe ( largely unaffected by 1 . 3 One - colour Custom Microarray Based Gene Expression AD pathology ) . A wealth of clinical information was avail - 25 Analysis ( Agilent ) able about each patient, including : Braak stage ( severity of 8x15K Custom Microarrays ( Agilent Technologies : able AD ) ; additional pathology ; age of onset and age at death ; to accommodate up to 15 ,000 genes ) were designed to personal and family history of cancer ; plasma homocysteine include various housekeeping genes ( 719 genes ) , internal levels , and the results of annual clinical tests including test controls ( 3141 genes ), and genes that were differentially of cognitive performance (CAMCOG ) . 30 expressed in AD brain relative to control brain based on a 1 . 2 DNA , RNA and Protein Extraction published dataset ( Xu , P . T . et al. Differences in apolipo RNA , DNA and protein were isolated from lateral tem - protein E3/ 3 and E4 / 4 allele - specific gene expression in poral, frontal and occipital lobe tissue of each patient by hippocampus in Alzheimer disease . Neurobiol. Dis . 21 , TRI- reagent extraction . 100 mg of frozen tissue was 256 - 275 ( 2006 ) ; Xu , P . T . et al. A SAGE study of apolipo homogenised in 1 ml TRI- reagent , incubated at room tem - 35 protein E3 / 3 , E3 /4 and E4 / 4 allele - specific gene expression perature (RT ) for 5 minutes , and supplemented with 100 ul in hippocampus in Alzheimer disease . Mol. Cell Neurosci . 1 - bromo - 3 - chloropropane . The solution was vigorously 36 , 313 - 331 ( 2007 ) (3718 genes ) . The remaining 7422 mixed for 15 seconds , incubated for 2 minutes , and centri - spaces were filled with known rapamycin - regulated genes fuged at 12 ,000 rotational centrifugal force ( g ) for 15 (based on an IPA Ingenuity search ) and the genes that were minutes at 4° C . to separate the RNA , DNA and protein 40 identified as rapamycin - regulated in lymphocytes based on layers. two -colour microarray based gene expression analysis . The aqueous RNA layer was transferred to an eppendorf, Of the 252 patients for whom tissue was available through supplemented with 500 ul isopropanol and mixed gently by the Thomas Willis brain bank , 32 patients were selected for inversion . After five minute incubation , the solution was one -colour custom microarray based gene expression analy centrifuged at 12 , 000 g for 8 minutes at 4° C . The pellet was 45 sis . They included control subjects and patients with mild washed in 1 ml 75 % ethanol, centrifuged at 7 , 500 g for 5 and advanced AD . The exclusion criteria were as follows : minutes at 4° C . , and ethanol wash removed . The pellet was vascular disease ; Parkinson ' s disease ; ApoE €2 / €2 , ApoE air -dried for 30 minutes and rehydrated in 100 ul nuclease €2/ €3 and ApoE €4 / €4 genotypes ( not enough patients for free water by incubation at 55° C . for 15 minutes, prior to meaningful statistical analysis ) ; and high plasma homocys storage at - 80° C . 50 teine level ( > 35 UM ) . RNA was converted to cDNA by reverse transcription : 50 1 . 3 . 1 Sample Preparation ul of 200 ng /ul RNA was added to 50 ul of Reverse RNA was extracted from the frontal lobe of each subject Transcriptase master mix (composed of 10 ul 10x reverse by TRI- reagent protocol as described in 1 .2 . The RNA was transcriptase buffer; 4 ul dNTP mix ( 100 mM ) ; 10 ul random treated with DNase . The suitability of the RNA for microar primers ; 5 ul MultiScribe Reverse Transcriptase (50 U / ul) ; 55 ray based gene expression analysis was determined by and 21 ul nuclease free water ), and incubated at 25° C . for Agilent RNA nano - chip analysis . RIN values ranged from 10 minutes ; 37° C . for 120 minutes and 85° C . for 5 seconds 2 - 3 indicating relatively poor quality RNA . However , this in a Thermal cycler. The cDNA was precipitated with was unavoidable , as the source brain tissue had significant isopropanol ( 100 ul cDNA supplemented with 20 ul of 3M post -mortem time prior to freezing, resulting in inevitable sodium citrate at pH 5 and 400 ul isopropanol) , centrifuged 60 degradation . at high speed for 10 minutes , and the resulting pellet washed 1 . 3 . 2 Conversion of RNA to labelled cRNA in ice cold 100 % ethanol. Following high speed centrifuga - 200 ng of RNA was converted to cDNA , and subsequently tion and supernatant removal , the pellet was air dried to labelled cRNA , with the low - input quick amplification overnight, and rehydrated in 100 ul nuclease free water , labelling kit. The spike mix was incubated at 37° C . for 5 prior to storage at 4° C . 65 minutes , and diluted in the provided dilution buffer ( Agilent The remaining TRI- reagent layers were supplemented Techologies ) as shown in Table 6 . 2 ul of the diluted spike with 300 ul ethanol and centrifuged at 12 ,000 g for 5 minutes mix was added to the RNA (200 ng ) in a 1. 5 ul volume. US 9 ,944 ,986 B2 79 80 Cyanine 3 -CTP was used to label all samples . The labelled TABLE 8 - continued and amplified cRNA samples were purified by standard Qiagen RNeasy mini column protocol, and quality assessed Microarray scanner settings for one - colour microarray and quantified . based gene expression analysis (Agilent ) 5 TABLE 6 8 x 15K HD microarray format Scan resolution (UM ) Preparation of Spike Mix Tiff 20 bit 10 - Spike mix Starting amount of RNA volume 1 . 3 . 4 . Data Analysis The feature extraction programmewas used to collate the Total per 15 Custom Microarray layout with the output of the scanner . RNA Concentration Serial dilutions labelling The results from individual patients were grouped based on subject diagnosis , disease severity ( as defined by Braak ( ng) ( ng/ ul ) First Second Third Fourth (ul ) stage ) and ApoE genotype . For the purposes of this study , the groups - of- interest were as follows ( see Table 9 ): 200 133 . 3 1 : 20 1 : 25 1 :10 2 20 1: 10 220 TABLE 9 1 .3 .3 Hybridisation Patient diagnosis ApoE genotype Number of patients Control ( entorhinal stage ) ApoE €3/ 83 600 ng of labelled CRNA was added to various fragmen - 25 MildM AD ( limbic age ) ApoE £3 /83 tation components ( Table 7 ) , incubated at 60° C . for 30 Advanced AD (neocortical stage ) ApoE £3/ 83 Auu minutes, and cooled on ice for 1 min . 25 ul of GEX Advanced AD ( neocortical stage ) ApoE 83/ 84 Hybridisation buffer HI- RPM was added to stop fragmen tation . The sample was gently mixed , centrifuged at 13 , 000 0 Statistical Analysis of Microarray (SAM ) was used to rpm for 1 min , and placed on ice in preparation for hybri - 30 carry out unpaired , two -sample T tests for each gene in a disation . The microarray was assembled and incubated over group -of - interest compared to control. SAM identifies genes night: the Custom 8x15K Microarray was used and 40 ul of that are differentially expressed at RNA level in the group sample was added per 15K array . After 17 hour hybridisa of- interest compared to control group ; the direction of tion , the microarrays were washed and scanned on the 35 expression ; fold change; and an estimate of the false dis Agilent C Scanner on programme AgilentHD _ GX _ 1 color, covery rate (FDR ). For the purpose of our study, we selected with the settings amended as shown in Table 8 . an estimated FDR of 10 % as acceptable for identifying differentially expressed genes . SAM was carried out with TABLE 7 1000 permutations, and the output processed to remove 40 duplicates . The output was analysed with the IPA Ingenuity Fragmentation components software (www .ingenuity . com ). Tables 2 shows differentially expressed transcripts in Volume/ Mass ( for 8 x 15K brain from early AD patients ( limbic stage ) relative to Components ( Agilent ) microarray ) 45 control (entorhinal stage ). Table 3 shows differentially expressed transcripts in brain from advanced AD patients Cy3- labelled CRNA 600 ng (neocortical stage ) relative to control (entorhinal stage ). 10 x blocking agent 5 ul Nuclease free water Bring total volume to 24 ul Example 2 25 x fragmentation buffer 1 ul 50 Total volume 25 ul Q -PCR Validation of Microarray Results Real -time PCR ( Q - PCR ) allows relative quantification of 55 a gene- of- interest by calculating the expression of the gene relative to a housekeeping gene such as beta -actin , allowing TABLE 8 patient to patient comparison . The validation study was carried out on cDNA obtained from the same patients and Microarray scanner settings for one - colour microarray brain regions that were used for the microarray study . The based gene expression analysis (Agilent ) genes were selected as they were shown to be either sig nificantly up - or down -regulated in advanced AD (neocor 8 x 15K HD microarray format tical stage ) compared to control in the microarray study. The Universal probe library design centre (Roche Diagnostic Dye Channel Green 65 Website ) was used to design Q -PCR systems for the genes Scan region Scan area (61 x 21 . 6 mm ) ( see Table 10 ); and primers and probes ordered from Sigma Genosys and Roche respectively . US 9, 944 ,986 B2 81 TABLE 10 Roche probe , primer sequences , and optimal annealing temperature corresponding to each gene - of - interest Optimal Forward Backward annealing Roche primer primer temperature Gene Probe ( 5 ' - 3 ' ) ( 5 ' - 3 ' ) ( ° C . ) Beta actin 24 TCAGCTGTGGGGTC GAAGGGGACAGGCAG 62 CTGT TGAG ( SEQ ID NO : 1 ) ( SEQ ID NO : 2 ) EIF4E 35 GATGGCGACTGTCG TGGGTTAGCAACCTC 60 ( Variant AAcc CTGAT 1 and 2 ) ( SEQ ID NO : 3 ) ( SEQ ID NO : 4 )
EIF4E 3535 GTGTAGCGCACACT TGGGTTAGCAAccTC 60 ( Variant TTCTGG CTGAT 3 ) ( SEQ ID NO : 5 ) ( SEQ ID NO : 6 ) MAPK1 62 CCGTGACCTCAAGC GCCAGGCCAAAGTCA 58 ( Variant ???c CAG 1 and 2 ) ( SEQ ID NO : 7 ) ( SEQ ID NO : 8 ) GABBR2 GCGAAGGACAGTGG GAGAGGGCGGATGGA 62 AGAAGT GATA ( SEO ID NO : 9 ) ( SEO ID NO : 10 ) SEMA4C 14 TTGTGCCGCGTAAG CAGCGTCAGTGTCAG 60 ACAGT GAAGT ( SEQ ID NO : 11 ) ( SEQ ID NO : 12 ) DZIP3 39 TGC?CAAGATCTGA ??????????????? 60 TACAAGG GTACA ( SEQ ID NO : 13 ) ( SEQ ID NO : 14 ) SERPINE1 80 CTCCTGGTTCTGCC CAGGTTCTCTAGGGG 58 CAAGT ???cc ( SEQ ID NO : 15 ) ( SEQ ID NO : 16 ) 35 For the composition of each 20 ul Q -PCR mix see Table curve copy number varied less than 10 % from the assigned 11 . Two negative controls (water ) and a cDNA standard copy number . The values obtained for each gene were curve ( five serial dilutions starting with neat cDNA ) were normalised to the corresponding beta -actin values to allow included per Q - PCR run . The samples were denatured at 96º . quantitative comparison of samples . C . for 15 minutes and amplified by 40 cycles of 96° C . for 15 seconds , optimal annealing temperature ( Table 10 ) for 30 Example 2 seconds and 72° C . for 30 seconds. FAM output was read in the annealing phase . Modulation of Rapamycin - sensitive Genes has the 45 Same Beneficial Effect on Alzheimer' S TABLE 11 Disease -related Protein Expression as Rapamycin Composition of Q -PCR reaction Manufacturer 2 . 1 Methods 0 . 5 ul Universal probe ( 10 UM ) Roche The genes identified as rapamycin -sensitive genes (exist 10 ul Absolute Q -PCR mix ThermoScientific (Compositition : 0 .625 Units ThermoPrime Taq 50 ing Table 1 ) were used for computer based in silico ) DNA polymerase, 75 mM Tris HCl (pH 8 . 8 at molecular network modelling and analysis ( using the IPA 25° C . ) , 20 mM (NH4 ) 2SO4 , 1 . 5 mM MgCl2, molecular network tools ) . In silico simulation ofmolecular 0 .01 % ( v / v ) tween 20 , 0 . 2 mM each of dATP , dCTP , dGTP, DTTP ) . interactions in the AD brain was also carried out based on 0 . 5 ul forward primer (20 uM ) Sigma Genosys the expression pattern of the rapamycin - sensitive genes 0 . 5 ul backward primer ( 20 UM ) Sigma Genosys 55 shown in Table 2 . Selected rapamycin - sensitive genes were 6 . 5 ul nuclease free water Qiagen used for further simulations to predict the effect of silencing 2 ul cDNA (neat , 1 : 4 and 1 :16 ) Prepared as these rapamycin - sensitive genes on AD - related pathology . above The simulations were followed by experiments to verify whether the modulation of the downstream effectors of 2 . 1 Data Analysis 60 mTOR ( rapamycin - sensitive genes ) would lead to measure Each of the cDNA standard curve serial dilutions were able changes in MAPT (microtubule associated protein tau ) assigned an arbitrary copy number ( 1 : 1 = 10 ,000 ; 1 : 2 = 5 ,000 ; similar to rapamycin . 1 :4 = 2500 ; 1 :8 = 1250 ; 1: 16 = 625 ) . The Rotor gene -6 pro In the cellular models used , the mTOR activation is gramme automatically identifies the optimal threshold and achieved by growth factors in the serum . The inhibition of determines the copy number of the gene - of - interest relative 65 mTOR by the addition of rapamycin counteracts this effect to the standard curve for each sample . The Q - PCR was and reduces the production of AD - type phospho - tau in the considered fully optimised when the calculated standard cultures . US 9 ,944 ,986 B2 83 84 Cell Culture intensity per cell ( for positive cells only ) were analysed SH -SY5Y human neuroblastoma cells were purchased separately . Additionally the proportion of positive cells in from ECACC and cultured in DMEM /F - 12 (Sigma ) supple each population was calculated . mented with 10 % FCS Gold ( PAA ), 100 U penicillin - 2 . 2 Results streptomycin ( Invitrogen ) and 2 mM L - glutamine (Sigma ). 5 2 .2 . 1 the Effects of Rapamycin Cells were kept in a humidified atmosphere at 37° C . and 5 % The in silico simulations ( using the IPA molecular net CO , . Cells were seeded in 96 -well plates and cultured for 24 work modelling tool) indicated that the rapamycin regulated hours before siRNA treatment. molecules interact with the AD - related proteins APP (amy siRNA Treatment of SH - SY5Y Cells loid precursor protein ) and the microtubule associated pro siRNA was purchased from Origene and applied at 1 nM 10 tein tau (MAPT ) . The in silico analysis indicated that the concentration for 48 hours . siRNA duplexes (Origene ) were activation ofmTOR in normal circumstances would lead to supplied as 20 uM stock solutions. siRNA was diluted in the inhibition of MAPT and APP . OPTIMEM to a 300 nM concentration and incubated at The gene expression patterns of rapamycin -sensitive room temperature (RT ) for 10 min . Lipofectamine genes in early stage AD patients ( as shown in existing Table (RNAIMAX ) was also diluted in OPTIMEM and incubated 15 2 ) were used to predict the activation state of APP and at RT for 10 min . The lipofectamine mix and the duplex mix MAPT in the brain . Based on the expression pattern of were added to the culture medium ( antibiotic free ) to rapamycin - sensitive genes in the early ( limbic ) stage of AD , achieve the final concentration of 1 nM siRNA and 0 . 3 % the inhibition ofMAPT and APP was predicted . The in silico lipofectamine . Cells were incubated at 37° C . for 4 hours, simulation also predicted that this gene expression pattern is and then media was replaced with antibiotic free media until 20 associated with the activation of mTOR . However, the collection . Some cultures were treated with additional expression pattern of many rapamycin - sensitive genes in the rapamycin for the last 24 hours of the culture period . brain of AD patients is inconsistent with the known normal Immunostaining molecular interactions. This indicates that the AD - related Cells were sequentially fixed in Glyo - Fixx ( Thermo Sci - deregulation of the mTOR pathway is also associated with entific ) for 2 hours atRT and in 85 % cold ethanol for 30 min . 25 unexpected variations from normal molecular interactions Blocking ( of non - specific staining ) was performed for 30 that are compatible with the idea that these molecules have min at RT using 5 % BSA and 0 . 1 % Triton - X - 100 in PBS . variants ( SNPs or other genetic variations ) that interfere Cells were incubated with primary antibody overnight at 4° with their normal interactions. C . ( for the negative controls , cells were incubated with The simulated inhibition of mTOR in this system ( using PBS - Triton only ) . Cells were washed in PBS - 0 . 1 % Triton 30 rapamycin ) will reverse the AD - associated inhibition of and incubated with secondary antibody (FITC conjugated ) MAPT and APP , indicating that rapamycin could reverse the for 2 hours at 4° C . Cells were washed in PBS -0 . 1 % Triton molecular expression changes seen in AD . and propidium iodide counterstained . The antibodies used in the experimental paradigm mTOR is activated in the were mouse polyclonal to phospho - Tau ( Abcam , 1 :200 ) and neuronal cells ( in the presence of growth factors from the anti -mouse IgG - FITC ( Abcam , 1 :400 ) . 35 serum ) . The addition of rapamycin ( 100 ng /ml ) will inhibit Propidium Iodide Staining mTOR , leading to the down regulation of AD - related phos Cells were incubated with propidium iodide ( Invitrogen , pho - tau ( p - tau ) in the cells . 10 ug /ml ) supplemented with RNase A (Sigma , 100 ug /ml ) In the SH -SY5Y cellular model, p - tau expression was for 20 min at 37° C . and scanned . regulated in a cell - cycle dependent manner and protein Cytometry 40 content of cells was generally higher in the cells that were Cytometry was performed using the Acumen Explorer in the G2 phase of the cell cycle relative to cells in the G1 TTP Lab Tech , Ltd . ( Software version 3 . 1 . 12 ) . phase of the cell cycle (FIG . 1 : White bars represent the G1 The propidium iodide staining was used to determine the phase cell population ; black bars represent the G2 phase cell cycle phase of the cells based on DNA content. It was population ). measured using a 488 nm excitation laser triggering the 3° 45 The effects of mTOR inhibition by rapamycin had the channel (bandpass filter 585 -620 nm ) . following effects . Firstly , the cell cycle kinetics were altered The immunostaining was used to measure the content of such that the G1 phase became longer and the G2 phase was phospho - Tau in the cells. Measurement was carried out shortened , as reflected by the accumulation of cells in the G1 using a laser triggering the 1° channel (bandpass filter phase of the cell cycle at the expense of the G2 phase ( FIG . 500 -530 nm ) . 50 2 : Vertically shaded bars represent the G1 population ; the Cell Cycle Analysis horizontally shaded bars represent the G2 cell population . To determine cut- offs for cells in different phases of the Lighter shades represent cells treated with Culture medium cell cycle , gate setting was performed based on the G1 and alone . Darker shades represent cells treated with Culture G2 peaks on the DNA content histogram . To determine the medium containing 100 ng /ml rapamycin . All data is nor G1 and G2 peaks, the 3° total intensity in 20 % histograms 55 malised to Control ( 100 % )) . Secondly , the rapamycin was analysed for each plate . Gates calculated were manually induced a reduction of p -tau content in the whole cell entered into the Acumen software . culture . This is partly due to the alterations in cell cycle The data exported and quantified included separately all kinetics ( reduction of the G2 cell population with the cells and single cells . Single cells were further subcatego generally higher p - tau content) . However, the rapamycin rised into euploid , apoptotic and polyploid cells. Euploid 60 also had a cell cycle independent effect on p - tau leading to cells were additionally classified into cells in G1S and G2M further reductions in this protein (FIG . 3 : Grey bars repre phases of the cell cycle . sent all single cells ; Vertically shaded bars represent the G1 Cellular Protein Measurement population ; the horizontally shaded bars represent the G2 The total fluorescence intensity from the 1° channel was cell population . Lighter shades represent cells treated with used to compare total protein levels in the different cell 65 Culture medium alone. Darker shades represent cells treated populations defined above . Mean fluorescence intensity per with Culture medium containing 100 ng /ml rapamycin . All cell ( for the whole of the population ) and mean fluorescence data are normalised to Control ( 100 % ) ) . US 9 ,944 ,986 B2 85 86 These findings indicate , in accordance with previous The effect of GABBR2 knockdown on p -tau expression studies, that rapamycin is able to modulate the accumulation was similar to that induced by rapamycin , albeit weaker. of AD - type p - tau both in a cell cycle dependent and inde However , in the presence of rapamycin , GABBR2 knock pendent manner. down had no further significant effect on p - tau expression in The possibility that modulation of downstream effectors 5 cells (FIG . 7 : Grey bars represent all single cells ; vertically of mTOR (shown in Table 1 ) would have a similar effect to shaded bars represent the cell population in the G1 phase of that observed with rapamycin was subsequently investi the cell cycle ; horizontally shaded bars represent the cells in gated . the G2 phase of the cell cycle. Lighter shades represent cells treated with Culture medium , siRNA Control and GABBR2 2 . 2 . 2 CACNA1 D 10 siRNA alone. Darker shades represent cells treated with CACNA1D was not previously known to be a rapamycin additional 100 ng/ ml rapamycin . All data are normalised to sensitive gene . Subsequently , the in silico molecular simu Control (100 % )) . lation of CACNA1 D knockdown did not predict alteration The data provide evidence that , contrary to prior knowl in the expression ofMAPT or APP . edge , the GABBR2 gene expression is mTOR dependent. However , the experimental data showed that thene cell cycle 1515 The data also provide evidence that modulation ofGABBR2 effects of CACNA1 D knock - down by siRNA were similar has a similar effect to rapamycin in terms of cell cycle and to that of rapamycin ( FIG . 4 : Vertically shaded bars repre - AD -related p - tau expression . sent the cell population in the G1 phase of the cell cycle , and The data provides evidence that the modulation of a the G1 time; horizontally shaded bars represent the cells in downstream effector of mTOR has the samebeneficial effect the G2 phase of the cell cycle and the G2 time. Lighter 20 on AD - related cell cycle and protein changes as rapamycin . shades represent cells treated with Culture medium , siRNA 2 . 2 . 4 HOXD10 control and CACNA1 D siRNA alone . Darker shades rep - The HOXD10 gene was not previously known to be resent cells treated with an additional 100 ng/ ml rapamycin . rapmycin - sensitive . Thus the in silico simulation of All data is normalised to Control ( 100 % ) . The effect of HOXD10 knockdown did not predict a similar effect to CACNA1 D knock - down by siRNA did not alter the effect 25 rapamycin with respect to AD - related protein expression . of rapamycin . This is consistent with the cell cycle modu - In the experimentalmodel , the siRNA to HOX10D did not lator effect of CACNA1 D downstream of mTOR . affect the cell cycle in a similar fashion to rapamycin ( FIG . Protein expression analysis indicated that the effect of 8 : Vertically shaded bars represent the cell population in the CACNA1 D knockdown is similar to that induced by G1 phase of the cell cycle , and the G1 time; horizontally rapamycin . However , the CACNA1 D knockdown did not 30 shaded bars represent the cells in the G2 phase of the cell alter the effect of rapamycin on p - tau expression in cells cycle and the G2 time. Lighter shades represent cells treated (FIG . 5 : Grey bars represent all single cells ; Vertically with Culture medium , siRNA Control and HOX10D siRNA shaded bars represent the cell population in the G1 phase of alone . Darker shades represent cells treated with additional the cell cycle ; horizontally shaded bars represent the cells in 100 ng /ml rapamycin . All data are normalised to Control the G2 phase of the cell cycle . Lighter shades represent cells 35 ( 100 % ) ) . However, rapamycin was able to exert its cell cycle treated with Culture medium , siRNA Control and CACNA1 modulator effect even when HOX10D was not expressed D siRNA alone . Darker shades represent cells treated with (FIG . 8 ) . This would indicate that HOX10D does not play a additional 100 ng /ml rapamycin . All data are normalised to role in the rapamycin induced cell cycle modulation . Control ( 100 % ) ) . HOX10D knockdown had a weak effect in reducing p - tau The data provide evidence that , contrary to prior knowl- 40 expression in the cellular model , mainly by reducing the edge , the CACNA1 D gene expression is mTOR dependent. amount of p -tau in the G1 cell population . The effects were The data also provide evidence thatmodulation of CACNA1 significantly weaker than that of rapamycin and the D has a similar effect to rapamycin in terms of cell cycle and HOXD10 knockdown did not affect the rapamycin effect AD - related p - tau expression . ( FIG . 9 : Grey bars represent all single cells ; Vertically The data provide evidence that the modulation of a 45 shaded bars represent the cell population in the G1 phase of downstream effector of mTOR has the same beneficial effect the cell cycle ; horizontally shaded bars represent the cells in on AD - related cell cycle and protein changes as rapamycin . the G2 phase of the cell cycle . Lighter shades represent cells 2 . 2 . 3 GABBR2 treated with Culture medium , siRNA Control and HOX10D The GABBR2 receptor has not previously been identified siRNA alone . Darker shades represent cells treated with as a downstream effector of mTOR i . e . is not a known 50 additional 100 ng /ml rapamycin . All data are normalised to rapamycin - sensitive gene . Thus the in silico simulation of Control ( 100 % ) . The data provide evidence that the modu GABBR2 receptor knockdown did not predict a similar lation of a downstream effector of mTOR has the same effect to rapamycin with respect to AD -related protein beneficial effect on AD -related protein changes as rapamy expression . cin . However , the effects of GABBR2 knockdown by siRNA 55 2 . 2 . 5 KLF2 in the experimental model were similar to that of rapamycin . Although KLF2 was not previously identified as a The GABBR2 knockdown did not affect the rapamycin rapamycin - sensitive gene , the indirect molecular interac response significantly (FIG . 6 : Vertically shaded bars rep tions allowed the simulation of the effect of mTOR on resent the cell population in the G1 phase of the cell cycle , KLF2 . However , in AD the expression of KLF2 was found and the G1 time; horizontally shaded bars represent the cells 60 to be opposite to what would normally be expected in in the G2 phase of the cell cycle and the G2 time. Lighter response to mTOR activation . The effects of KLF2 knock shades represent cells treated with Culture medium , siRNA down in the AD brain were simulated , and found to closely Control and GABBR2 siRNA alone . Darker shades repre - mimic the effects of rapamycin in terms of AD -related sent cells treated with additional 100 ng /ml rapamycin . All protein expression (MAPT and APP ) . data is normalised to Control ( 100 % ) ) . These data are 65 In the experimental model , the knockdown of KLF2 consistent with the cell cycle modulator effect of GABBR2 produced similar but weaker cell cycle effects relative to downstream ofmTOR . rapamycin . The KLF2 knockdown did not affect signifi US 9 ,944 ,986 B2 87 88 cantly the effect of rapamycin on the G1 phase of the cell 2 .2 .7 GLI2 cycle . However , the KLF2 knockdown led to a significant Based on known molecular interactions it could be pre alteration of the G2 effect of rapamycin (FIG . 10 : Vertically dicted in silico that GLI2 (although not previously identified shaded bars represent the cell population in the G1 phase of as a rapamycin - sensitive gene ) would be differentially regu the cell cycle , and the G1 time; horizontally shaded bars 5 lated by mTOR activation . The AD brain expression studies represent the cells in the G2 phase of the cell cycle and the however indicated that the expression of GLI2 is not con G2 time. Lighter shades represent cells treated with Culture sistent with the activation of mTOR . The in silico simula medium , siRNA Control and KLF2 siRNA alone . Darker tions carried out to predict the effects of GLI2 knockdown in the AD brain indicated that the knockdown ofGLI2 would shades represent cells treated with additional 100 ng The/ml 10 lead to effects that are very similar to that of rapamycin in rapamycin . All data are normalised to Control ( 100 % ) ) . The termsof AD -related protein (MAPT and APP ) expression in data indicate that KLF2 is a downstream effector of the the brain . mTOR induced cell cycle response and it is essential for the The effects of GLI2 knockdown by siRNA on the cell G2 regulator effects ofmTOR . cycle were similar to that of rapamycin , but had no effect on The effects of KLF2 knockdown on p - tau expression were 1515 rapamycin response ( FIG . 14 : Vertically shaded bars repre similar to those of rapamycin , albeit a lot weaker . The KLF2 sent the cell population in the G1 phase of the cell cycle, and knockdown did not affect the rapamycin effect on p - tau the G1 time; horizontally shaded bars represent the cells in expression ( FIG . 11: Grey bars represent all single cells ; the G2 phase of the cell cycle and the G2 time. Lighter Vertically shaded bars represent the cell population in the 1 shades represent cells treated with Culture medium , siRNA phase of the cell cycle; horizontally shaded bars represent 20 Control and GLI2 siRNA alone . Darker shades represent the cells in the G2 phase of the cell cycle . Lighter shades cells treated with additional 100 ng /ml rapamycin . All data represent cells treated with Culture medium , siRNA Control are normalised to Control ( 100 % ) ) . and KLF2 siRNA alone . Darker shades represent cells The data indicate that in the absence of rapamycin the treated with additional 100 ng /ml rapamycin . All data are effect of GLI2 knockdown is similar to that induced by normalised to Control ( 100 % ) ) . 25 rapamycin , albeit weaker ( FIG . 15 : Grey bars represent all These data also indicate that the modulation of a rapamy single cells ; Vertically shaded bars represent the cell popu cin - sensitive gene will lead to effects that are similar to lation in the G1 phase of the cell cycle ; horizontally shaded rapamycin . bars represent the cells in the G2 phase of the cell cycle . 2 . 2 .6 RHO Lighter shades represent cells treated with Culture medium , RHO was not previously known to be a rapamycin - 30 siRNA Control and GLI2 siRNA alone . Darker shades sensitive gene . However , the molecular interactions of RHO represent cells treated with additional 100 ng /ml rapamycin . All data are normalised to Control ( 100 % ) ) . However , in the allowed the simulation of the effect of RHO knockdown in presence of rapamycin GLI2 knockdown had no further the AD brain . The in silico simulation indicated that RHO significant effect on p - tau expression in cells . knockdown will lead to changes in the AD -related molecules 3525 The data provide evidence that modulation of GLI2 has a (MAPT and APP ) similar to that seen with Rapamycin . similar effect to rapamycin in terms of cell cycle and In the experimental model the knockdown of RHO pro AD - related p -tau expression . duced similar , but weaker cell cycle effects relative to In summary , the modulation of individual rapamycin rapamycin . The RHO knockdown did not affect significantly sensitive genes can achieve the same beneficial effect on the effect of rapamycin on the G1 phase of the cell cycle . 40 neurones as rapamycin . However, the RHO knockdown led to a significant alteration of the G2 effect of rapamycin (FIG . 12 : Vertically shaded Example 3 bars represent the cell population in the G1 phase of the cell cycle , and the G1 time; horizontally shaded bars represent Modulation of mTOR Activity can be Detected by the cells in the G2 phase of the cell cycle and the G2 time. 45 Imaging Metabolic Markers Associated with Lighter shades represent cells treated with Culture medium , Rapamycin - sensitive Genes siRNA Control and RHO siRNA alone . Darker shades represent cells treated with additional 100 ng /ml rapamycin . 3 . 1 MRI All data are normalised to Control ( 100 % ) . The data To analyse the effect ofmTOR activation and inhibition indicate that RHO is a downstream effector of the mTOR 50 on the brain , SD rats ( 170 -220 g ) were treated with rapamy induced cell cycle response and it is essential for the G2 cin ( inhibitor of mTOR ; 0 . 2 mg/ kg i. p . ) and ketamine regulator effects of mTOR . (activator of mTOR ; 30 mg/ kg i. p ) and neuroimaging results The effects of RHO knockdown on p - tau expression were from these experiments were compared to control ( un similar to those of rapamycin , albeit a lot weaker. The RHO treated ) animals . There were three animals ( n = 3 ) in each knockdown did not affect the rapamycin effect on p - tau 55 group . Animals were sacrificed and the brain removed and expression ( FIG . 13 : Grey bars represent all single cells ; frozen for imaging studies. Vertically shaded bars represent the cell population in the G1 Whole organs were fixed in 0 . 154 M LiCl in 10 : 1 H , O : phase of the cell cycle ; horizontally shaded bars represent formaldehyde prior to MRI experiments . All imaging the cells in the G2 phase of the cell cycle . Lighter shades experiments were performed on a Bruker DMX300 spec represent cells treated with Culture medium , siRNA Control 60 trometer, at a ' H NMR resonance frequency of 300 MHz at and RHO siRNA alone. Darker shades represent cells treated 289 . 5 + 0 . 2 K . All images were acquired using a 30 mm with additional 100 ng/ ml rapamycin . All data are nor radiofrequency resonator. Images were acquired using a malised to Control ( 100 % ) ) . These data are consistent with spin - echo imaging technique [ 1 ] . A set of either 7 or 8 RHO acting downstream ofmTOR . equally spaced , coronal slices of 1 mm thickness , with a The data also indicate that the modulation of a rapamycin - 65 matrix size of 128x32 pixels and field -of - view of 30 mmx10 sensitive gene will lead to effects that are similar to rapamy- mm , were collected along the length of the brain . The cin . recovery time was 15 s, to ensure full T1 relaxation between US 9 ,944 ,986 B2 89 90 each acquisition . A T , map , for each coronal slice , was The clear differences in the MRI imaging as well as the produced by acquiring between 16 and 24 echo images and differences in the magnetic spectroscopy indicate that varying the echo time from a minimum value of 3 ms to a changes in brain metabolism induced by mTOR inhibition or maximum value of 80 ms. These echo images were then activation can clearly be identified using methods already fitted to Equation 1 , resulting in a T , value for each pixel in 5 used in human imaging technology . the coronal slice . Example 4
- 1 My = Moe Tž The Effect of Genetic Polymorphisms on the Rapamycin - regulated Genes is Associated with the where My is the signal intensity for each pixel at time t and Rapamycin Response in Human Lymphocytes from M , is the signal intensity at t = 0 . Individual Patients and the Diagnosis of AD A transverse T , map was acquired for each brain using the 15 method described for the coronal maps. Each image com 4 . 1 in Silico Data Mining prised 128x128 pixel array, with a field of view of 3 cmx3 Existing databases were analysed to identify the mTOR cm . A total of 24 echo images were acquired for each regulated genes on the chromosomal regions identified to be transverse T , map , with echo times from 3 - 80 ms. Each linked to AD ( linkage studies , AlzGene ). transverse slice was positioned in the centre of the brain . The AlzGene database identifies the following chromo FIGS. 16A - C show clear differences in the T2 weighted somal regions in linkage with AD ( Table 12 ) MRI images obtained from Rapamycin treated , ketamine treated and Control animals . TABLE 12 25 3 . 2 Magnetic Resonance Spectroscopy Imaging Location Hamshere et al. Butler et al . In silico simulations of the effects of mTOR activation ChromosomeChr (Mb ) ( 2007 ) LOD (Mb ) (2009 ) P - value and inhibition on the accumulation of choline and creatine have been carried out. These simulations indicated that 1p31. 1 -q31 . 1 83 - 185 0 .004 - 0 .05 mTOR modulation leads to significant changes in choline 30 3q12. 3 - q25 .31 103 - 173 0 .03 and creatine levels . This is consistent with the possibility of 6p21. 1 - 215 43 - 91 0 .02 imagingmTOR activation / inhibition in the brain using mag- 7pter -q21 . 11 0 - 78 0 . 008 - 0 .04 netic spectroscopy . 8p22 -p21 . 1 13 - 28 0 .001 Methods 9p22 . 3 -p13 . 3 20 - 35 1. 2 (~ 23 Mb ) 35 9q21. 31- 232 80 - 100 2 .5 ( ~ 101 Mb ) All MSI imaging experiments were performed on a 10p14 - 224 10 - 100 3 . 3 ( ~ 61 Mb ) Bruker ultrafleXtreme TOF / TOF mass spectrometer . For 17424 . 3- qter 67 -79 0 . 03 matrix evaluation , 4 serial sections of thickness 10 um from 19p13 . 3 - qter 8 - 54 2. 0 (~ 52 Mb) 0 .01 - 0 . 05 sham control brain S2 were acquired using a Leica CM 1850 Cryostat and subsequently thaw mounted onto an ITO 40 coated glass slide. Each section in turn was coated with 15 Linkage regions in this table are based on results of the mL of 20 mgmL - 1 of CHCA in CH ,OH , 0 . 1 % TFA ) using jointJo ! and meta - analyses of previously published genome wide linkage (GWL ) data (Hamshere , M . L ., P . A . Holmans , an artist airbrush while the remaining 2 sections were et al. ( 2007 ) . Genome- wide linkage analysis of 723 affected covered . Data were collected over the mass range m / z 45 relative pairs with late -onset Alzheimer' s disease . Hum Mol 60 - 1400 . MSI experiments of a single section from each Genet 16 ( 22 ) : 2703 -12 ; Butler , A . W ., M . Y . Ng , et al. group (Rapa , Ket and Control) were conducted . Data were ( 2009) . Meta - analysis of linkage studies for Alzheimer ' s collected over the mass range m /z 60 - 1400 . Images were disease -a web resource. Neurobiol. Aging 30 ( 7 ): 1037 -47 ). acquired with a pixel size of 100 umx100 um . 50 Genes on the regions identified by AlzGene to be in The data set contains numerous ion images which dem - linkage with AD were identified from the Ensembl database onstrate grey and white anatomical differences and clear (http :/ / www .ensembl . org ) . Variations on the genes in the differences induced by mTOR inhibition or activation (by region were identified using BioMart and the Ensembl rapamycin and ketamine respectively relative to control) . variation 72 database , the contents of which are incorporated These molecular species belong to the group of Phosphati - 55 herein in their entirety . The results indicate that 22. 17 % of dylcholines ( FIG . 17 . First row Rapamycin treated ; Second rapamycin -regulated genes were found on these AD - related row - Ketamine treated ; Third row - Control. Columns : 1 ) chromosomal regions. Interestingly enough a much smaller m /z 769, 2 ) m /z 868 , 3 ) m / z 866 , 4 ) m / z 752 , 5 ) m /z 844 , 6 ) number of genes were found to be related to AD in prior m / z 840 , 7 ) m / z 780 ) . studies (see Table 13 ) Additionally there is evidence that , similar to the simu SNPs selected for analysis are shown in Table 14 . Further lated models , mTOR modulation ( by rapamycin or Ket - details of each of these SNPs can be found in the NCBI amine ) leads to measureable changes of choline (FIG . 18A . database dbSNP ( http : // www .ncbi . nlm .nih . gov/ SNP /) . The First row - Rapamycin treated , Second row - Ketamine contents of the dbSNP database entries for each of the SNPs treated , Third row - Control ) and creatine (FIG . 18B . First 65 listed in Table 14 are expressly incorporated herein by row — Rapamycin treated , Second row — Ketamine treated , reference , in particular for the purposes of further defining Third row - Control) levels in the brain . the location and identity of the SNP. US 9 ,944 ,986 B2 91 92 TABLE 13 No of genes No of rapa- Genes in % genes % of Rapa associated % genes % of AD regulated region % of regulated by regulated with AD ( from associated associated No of genes genes on the whole Rapa in genes in this existing with AD in genes in this Region on region region genome region region databases ) this region region 1p31. 1 - q31. 1 1575 46 7 .88 2 . 92 4 . 38 0 . 83 3 . 19 3q12. 3 - 425 .31 640 34 3 . 20 5 . 31 3 . 24 0 . 78 1 . 23 6p21. 1 - q15 204 1 . 02 4 . 90 0 . 95 0 . 00 0 . 00 7pter- q21. 11 991 Oo 4 . 96 4 .04 3 . 81 0 . 71 1 .72 8p22- p21 . 1 167 0 . 84 *4 . 19 0 . 67 2 . 99 1 . 23 9p22. 3 -p13 . 3 275 1 . 38 2 . 18 0 . 57 0 . 73 0 . 49 9q21. 31 - 232 430 2 . 15 2 . 79 1 . 14 HARUNNOU 1 . 16 1 . 23 10p14 - 424 1072 5 . 36 3 .64 3 . 71 1 .03 2 .70 17q24 . 3 - qter 335 1 .68 5 .07 1 .62 0 . 30 0 . 25 19p13 . 3 - qter 1164 NEW 5 . 82 1 . 89 2 . 09 0 ..95 95 2 . 70 Total 6853 233 34 .27 3. 40 22 . 17 60 0 .88 14 .74
TABLE 14 SNPs selected for analysis Position on 1000 genomes Variation Chromosome Variant global MAF Associated gene Distance to Code Name Chromosome (bp ) Alleles (ALL ) with phenotype transcript pri rs798893 54793830 G / C 0 . 2921 LILRB2 , intronic 4072 pr10 rs725106 190347665 G / A 0 . 375 FAM5C intronic 99094 pr11 rs1341665 159691559 G / A 0 . 3814 CRP pr12 rs1359059 167234166 G / A 0 . 4725 POU2F1 intronic * 44038 pr13 rs1532278 27466315 T / C 0 . 2821 CLU 2420 pr14 rs1801274 161479745 A / G 0 . 4304 FCGR2A 395 pr15 rs20361082026 26663100 C / T 0 . 2688 ADRATA intronic * * 57433 pr2 rs811925 106547372 C / G 0 . 136 PRDM1, intronic * * * 212 pr3 rs883524 23194591 T / C 0 . 1401 LOXL2 intronic * * * 3583 pr4 rs 1065457 158324425 A / G 0 . 4469 CD1E intronic 646 Prs rs1148613 190337300 AC 0 . 3723 FAM5C intronic 106583 pro rs295 19816238 AC 0 .2596 LPL 4576 Pr7 rs290258 93555739 ANG 0 .2592 SYK pr8 rs365836 101809851 AG 0 . 2601 CUX1 117399 pr9 rs569214 OOOOHOOOOOAA 27487790 G / T 0 . 3539 CLU 3595 The SNPs selected were either SNPs on mTOR - sensitive 40 PCR mix was assembled as follows: genes or their direct upstream regulators ( POU2F1 is a transcriptional regulator of the mTOR genes A2M , CRP , P21 ex2 (Forward Primer ) 0 . 2 ul CSFIR , CYP2C9 , ESR1, GSTM3, ?L2 , IL6 , PRKAA2, P21 ex2' ( Reverse Primer ) 0 . 2 ul SPP1, TLR4 from Table 1 ; ADRA1A is a regulator ofmTOR 45 Nuclease free water 15 . 6 ul regulated genes : CDKN1B , EGR1, FGF7 , FN1, IL6 , JUN , 2 x Reddy Mix PCR master mix 20 ul LOX , NR4A1, NR4A2 from Table 1 ; LOXL2 is an upstream regulator of mTOR regulated genes: CDH1, FN1, MMP9 from Table 1 ; PRDM1 is the upstream regulator of mTOR The reagents were added in the volumes as stated above regulated genes: ESR1. IGHG1. IL10 . IL2. IL6 . MYC . 50 to give a 36 ul 'Master Mix ' . The Master Mix is dispensed RELN , SCGN from Table 1 ) . in 36 ul aliquots , to which is added 4 ul of test DNA . The Methods PCR was carried out as follows: 95° C . for 5 minutes , The rapamycin response in peripheral lymphocytes was followed by 40 cycles of 95° C . for 60 sec , 57° C . for 60 sec measured in 39 patients . The method has been described and 72° C . for 60 sec . previously ( Yates et al, Dysfunction of the mTOR pathway 55 Following the PCR 4 ul of each sample was denatured in is a risk factor for Alzheimer ' s disease , Acta Neuropatho - 12 ul SSCP denaturing solution ( 95 % Formamide , 10 mM logical Communications, 2013 ) . NaOH , 0 .01 % w / v xylene cyanole and 0 .01 % w / v bro Genomic DNA was extracted from the same lymphocyte mophenol blue ). Samples were denatured at 95° C . for 6 samples (using established protocols ) . Primers were minutes and placed on ice for at least 10 minutes . Gel designed for the SNPs in Table 13 using Primer3 tool. PCR 60 electrophoresis was performed using 3 % Metaphore agarose was carried out using the 2x Reddymix PCR master mix ( Thermo scientific , AB - 0575 / DC /LD / B ) with a final com + 0 . 5 % multipurpose agarose gels , at 400 V for 45 minutes. position of: 0 .625 units ThermoPrime Taq DNA polymerase , The SNPs on the samples lead to single stranded variants 75 mM Tris- HCl (pH 8 . 8 at 25° C .) , 20 mM (NH ), SO , that ran at different speeds on the gel ( SSCP analysis ). 1 .5Mm MgCl2 , 0 .01 % ( v /v ) Tween 20 , 0 . 2 mM each of 65 The variant with the lower frequency was regarded the DATP , dCTP , dGTP and dTTP , and precipitant and red dye minor allele and labelled “ 1 ” ; while the more frequent allele for electrophoresis . was labelled “ O ” . US 9 ,944 ,986 B2 93 94 4 . 2 Minor allele frequencies and the association of these TABLE 19 alleles with AD . As the Chi- squared analyses indicated , none of the SNPs investigated was significantly associated on their own with Frequency table & Chi- squared test the diagnosis of AD ( see Tables 15 - 29 ) . 5 Codes X pr13 TABLE 15 Codes Y DG Codes X Frequency table & Chi- squared test Codes Y Codes X pr1 10 CONTROL 20 (51 . 3 % ) Codes Y DG Codes X PROB . AD 8 19 (48 . 7 % ) Codes Y CONTROL 13 20 (51 . 3 % ) 39 PROB . AD 14 19 (48 . 7 % ) 24 (61 . 5 % ) 15 (38 . 5 % ) 39 15 Chi- squared 0 .016 27 (69 . 2 % ) 12 ( 30 . 8 % ) 39 Chi- squared 0 . 058 DF DF Significance level P = 0 . 8992 Significance level P = 0 .8101 Contingency coefficient 0 .038 Contingency coefficient 0 .020 20
TABLE 16 TABLE 20 Frequency table & Chi -squared test Frequency table & Chi- squared test 25 Codes X pr10 Codes X pr14 Codes Y DG Codes Y DG Codes X Codes X Codes Y Codes Y 0 CONTROL ????? 20 (51 . 3 % ) CONTROL 15 20 (51 . 3 % ) PROB . AD ? 13 19 (48 . 7 % ) 30 PROB . AD 15 Au 19 (48 . 7 % ) 30 ( 76 . 9 % ) 9 ( 23 . 1 % ) 3939 30 (76 . 9 % ) 9 (23 . 1 % ) 39 Chi -squared 0 .719 Chi- squared 0 .008 DF DF Significance level P = 0 . 3964 Significance level P = 0 .9301 Contingency coefficient 0 . 135 Contingency coefficient 0 .014 35
TABLE 17 TABLE 21
Frequency table & Chi- squared test 40 Frequency table & Chi- squared test Codes X pr11 Codes X pr15 Codes Y DG Codes Y DG Codes X Codes X Codes Y 0 Codes Y 0 CONTROL 14 20 (51 . 3 % ) CONTROL 12 20 (51 . 3 % ) PROB . AD 13 aa 19 ( 48 . 7 % ) 45 PROB . AD 12 VO 19 (48 . 7 % ) 27 (69 . 2 % ) 12 ( 30 . 8 % ) 39 24 (61 . 5 % ) 15 (38 . 5 % ) 39 Chi -squared 0 . 058 Chi- squared 0 . 016 DF DF Significance level P = 0 . 8101 Significance level P = 0 .8992 Contingency coefficient 0 . 038 50 Contingency coefficient 0 .020
TABLE 18 TABLE 22 Frequency table & Chi- squared test Frequency table & Chi -squared test Codes X pr12 Codes X pr2 Codes Y DG Codes Y DG Codes X Codes X Codes Y O Codes Y 0 CONTROL 15 20 (51 . 3 % ) CONTROL 16 20 (51 . 3 % ) PROB . AD 13 ?? 19 ( 48 . 7 % ) 60 PROB . AD TN 19 ( 48 . 7 % ) 28 (71 . 8 % ) 1111 (2028 . 2 % ) 39 33 (84 .6 % ) 6 ( 15 . 4 % ) 39 Chi- squared 0 .010 Chi- squared 0 . 141 DF DF Significance level P = 0 . 9200 Significance level P = 0 . 7072 Contingency coefficient 0 .016 65 Contingency coefficient 0 . 060 US 9 ,944 , 986 B2 95 96 TABLE 23 TABLE 27 Frequency table & Chi- squared test Frequency table & Chi -squared test Codes X pr7 5 Codes Y DG Codes X pr3 Codes X Codes Y DG Codes Y o CONTROL ????? Codes X 17 20 (51 . 3 % ) PROB . AD 14 ? 19 ( 48 . 7 % ) Codes Y 31 ( 79. 5 % ) 8 ( 20 . 5 % ) 39 CONTROL 20 (51 . 3 % ) 10 Chi- squared 0 . 229 PROB . AD ose 65 19 (48 . 7 % ) DF 1 Significance level P = 0 .6326 Contingency coefficient 0 . 076 20 (51 . 3 % ) 19 (48 . 7 % ) 39 Chi- squared 0 .024 15 TABLE 28 DF Frequency table & Chi- squared test Significance level P = 0 . 8759 Codes X pr8 Contingency coefficient 0 .025 Codes Y ?G Codes X 20 Codes Y CONTROL 14 20 (51 . 3 % ) TABLE 24 PROB . AD 15 19 ( 48 . 7 % ) 29 (74 . 4 % ) 10 ( 25. 6 % ) 39 Frequency table & Chi- squared test Chi- squared 0 . 074 25 pr4 DF Codes X P = 0 . 7850 Codes Y DG Significance level Codes X Contingency coefficient 0 . 044 Codes Y CONTROL 20 (51 . 3 % ) PROB . AD AO 19 (48 . 7 % ) ao 30 TABLE 29 35 (89 . 7 % ) 4 (10 . 3 % ) 3939 Chi - squared 2 .683 Frequency table & Chi- squared test DF Codes X prg Significance level P = 0 . 1014 Codes Y DG Contingency coefficient 0 . 254 35 Codes X Codes Y O CONTROL 16 20 (51 . 3 % ) PROB . AD 16 WA 19 (48 . 7 % ) TABLE 25 32 (82 . 1 % ) 7 (17 . 9 % ) 39 Frequency table & Chi- squared test Chi- squared 0 .006 40 DF Codes X prs Significance level P = 0 . 9403 Codes Y DG Contingency coefficient 0 . 012 Codes X Codes Y CONTROL 16 20 (51 . 3 % ) However , when combinations of SNPs were investigated , PROB . AD 14 19 (48 . 7 % ) 45 it was found that different combinations of SNPs were UA significantly associated with the rapamycin response of the 30 (76 . 9 % ) 9 (23 . 1 % ) 39 individual patient as measured from lymphocytes . Chi -squared 0 . 008 DF The increase in cell death elicited by rapamycin in the Significance level P = 0 . 9301 lymphocytes was significantly associated with the combi Contingency coefficient 0 .014 50 nation of pr4 , prl and pr15 ( Table 30 ). TABLE 30 TABLE 26 Multiple regression Frequency table & Chi- squared test 55 Dependent Y f _ DEAD _ Rapa/ f _ DEAD _ Control Sample size 39 Codes X pro Coefficient of 0 . 2305 Codes Y DG determination RP Codes X Codes Y 1 Regression Equation CONTROL 11 20 (51 . 3 % ) PROB . AD FOO 8 19 ( 48 . 7 % ) 60 Independent variables Coefficient Std . Error (partial t P 20 (51 . 3 % ) 19 (48 . 7 % ) 39 Chi- squared 0 .235 ( Constant) 1 .0999 DF pr4 0 . 7790 0 .4291 0 .2933 1 .815 0 . 0781 Significance level P = 0 .6278 pr1 - 0 . 7175 0 . 3987 - 0 .2910 - 1 .800 0 . 0805 Contingency coefficient 0 .077 65 pr15 0 . 8547 0 . 3871 0 .3497 2 . 208 0 . 0339 F - ratio 3 . 4942 US 9, 944 ,986 B2 97 98 TABLE 30 -continued TABLE 33 Significance P = 0. 026 Multiple regression level Dependent Y difference between population 5 doubling level induced by Rapamycin The relative lengthening of the G1 time induced by Sample size 39 rapamycin in the lymphocyte cultures ( as defined in Zs Coefficient of 0 .5954 Nagy , M Combrinck , M Budge , R McShane. Cell cycle determination RP kinesis in lymphocytes in the diagnosis of Alzheimer ' s Regression Equation disease . Neurosci Letters . 2002 , 317 , 2 , 81 -84 ) was signifi- 10 cantly associated with a combination of pr4 , pr5 , pr7 , pr10 Independent and pr11 ( Table 31) . variables Coefficient Std . Error partial t P (Constant ) 1 . 1601 TABLE 31 pr12 - 0 . 5319 0 . 1663 - 0 . 4982 - 3 . 199 0 . 0032 15 pr3 - 0 . 4043 0 . 1423 - 0 .4544 - 2 . 840 0 .0079 Multiple regression Pr8 0 . 3558 0 . 1715 0 . 3492 2 . 075 0 . 0464 pr10 0 . 4772 0 . 1190 0 . 5844 4 .010 0 . 0004 Dependent Y Relative lengthening of G1 time pr11 - 0 . 3726 0 . 1756 - 0 . 3562 - 2 . 122 0 . 0419 Sample size 39 pr15 - 0 .2380 0 . 1250 - 0 . 3235 - 1 . 904 0 . 0663 Coefficient of 0 ..4594 LHcy - 0 .03578 0 .01574 - 0 . 3779 - 2 . 272 0 .0301 determination R ? F - ratio 6 . 5158 20 Significance P < 0 .001 Regression Equation level Independent variables Coefficient Std . Error Ipartial t P The change in the G1 time induced by Rapamycin in lymphocyte cultures was significantly associated with pr5 , (Constant ) 0 . 9648 25 pr4 0 . 3609 0 . 1148 0 .4802 3 . 145 0 . 0035 w pr7 , pr10 , pr11 and pr15 ( Table 34 ). Pr5 - 0 .3738 0 . 1476 - 0 .4033 - 2 . 532 0 .0163 pr7 0 . 2966 0 . 1120 0 . 4187 2 .649 0 . 0123 TABLE 34 pr10 0 .2171 0 .08211 0 .4182 2 .644 0 . 0124 pr11 0 . 3363 0 . 1350 0 . 3979 2 .492 0. 0179 Multiple regression F -ratio 5 .6095 30 Significance P = 0 .001 Dependent Y avg _ TG1' /avg _ TG1 level Sample size 39 Coefficient of 0 .4968 The difference between population doubling level determination R ? induced by Rapamycin in the lymphocyte cultures was 35 – Regression Equation significantly associated with the combination of pr4 , pró , Independent pr12 , pr13, prl, pr8 , pr10 and pr11 ( Table 32 ). variables Coefficient Std . Error ['partial t P TABLE 32 ( Constant) 1 . 2308 pr5 - 0 .8739 0 . 1888 - 0 .6274 - 4 .628 0 . 0001 Multiple regression 40 Pr7 0 . 2405 0 . 1438 0 . 2796 1 .673 0 . 1038 pr10 0 . 3729 0 . 1063 0 . 5212 3 .508 0 . 0013 Dependent Y difference between population pr11 0 .5171 0 . 1789 0 . 4495 2 . 891 0 . 0068 doubling level induced by Rapamycin pr15 - 0 . 2051 0 .1068 - 0 .3171 - 1 . 921 0 . 0634 Sample size 39 F - ratio 6 . 5156 Coefficient of 0 . 5932 Significance P < 0 .001 determination R ? 45 level Regression Equation The significant association between the SNPs on rapamy Independent cin - sensitive genes and the change in the G1 time induced by variables Coefficient Std . Error partial t P rapamycin in lymphocyte cultures was altered by plasma ( Constant ) 0 . 7941 homocysteine levels ( Table 35 ) . pr4 - 0 . 3640 0 . 1715 - 0 .3614 - 2. 123 0 .0421 pro - 0 .2529 0 . 1572 - 0 .2818 - 1. 609 0 . 1182 TABLE 35 pr12 - 0 . 6737 0 . 1633 - 0 .6016 - 4 . 125 0 .0003 pr13 - 0 . 2117 0 . 1259 - 0 .2936 - 1 .682 0 . 1029 Dependent Y avg_ TG1' / avg _ TG1 prl - 0 . 2015 0 . 1414 - 0 .2517 - 1 .424 0 . 1646 55 Sample size 39 pr8 0 . 3235 0 . 1766 0 .3172 1 .832 0 .0769 Coefficient of 0 .5859 pr10 0 .4391 0 . 1160 0 .5684 3 .784 0 . 0007 determination R2 pr11 - 0 .2514 0 . 1759 - 0 .2524 - 1 . 429 0 .1633 F -ratio 5 . 4687 Regression Equation Significance P < 0 . 001 level Independent 60 variables Coefficient Std . Error (partial t This association was significantly affected by the plasma (Constant ) 1 .6011 homocysteine levels of the patients measured at the time of pr5 - 0 . 7138 0 . 1943 - 0 . 5569 - 3 .673 0 .0009 pr7 0 . 2224 0 . 1432 0 . 2727 1 . 552 0 . 1311 the blood sample collection . Plasma homocysteine is an pr13 0 . 2103 0 . 1296 0 .2840 1 . 623 0 . 1151 independent environmental risk factor of AD and it is also 65 pr3 - 0 . 2627 0 . 1554 - 0 . 2949 - 1 .691 0 . 1013 known to affect cell proliferation and cell cycle control pr10 0 . 3731 0 . 1071 0 . 5365 3 . 482 0 .0015 functions ( Table 33 ) . US 9, 944 ,986 B2 99 100 TABLE 35 - continued nation of SNPs on rapamycin - sensitive genes (pr4 , pr5, pr7 , pr11 0 . 3414 0 . 1894 0 . 3126 1 .803 0 . 0815 pr10 , pr11, pr15 ) ( Table 38 ) and the association was sig pr15 - 0 . 1511 0 . 1129 - 0 .2374 - 1 . 338 0 . 1908 nificantly affected by plasma homocysteine levels ( Table LHcy - 0 . 02881 0 .01399 - 0 . 3519 - 2 .059 0 .0483 39) . F - ratio 5 . 3058 5 Significance P < 0 . 001 level TABLE 38 Multiple regression The baseline proliferation speed (population doubling time PDT) of the lymphocytes from individual patients also 10 Dependent Y avg _ PDT' _ PDT depended on a combination of SNPs (pr4 , pr5 , pr6 , pr12 , avg PDT /PDT pr13 , pr14 , prl, pr11 and pr15 ) ( Table 36 ) . Sample size 39 Coefficient of 0 . 4664 TABLE 36 determination R2 15 Multiple regression Regression Equation Dependent Y avg _ PDT avg PDT Independent Sample size 39 variables Coefficient Std . Error ( partial t P Coefficient of 0 . 6676 20 determination R ? (Constant ) 1 . 2206 Regression Equation pr4 - 0 . 1752 0 . 1156 - 0 . 2587 - 1 . 515 0 . 1396 Prs - 0 .6109 0 . 1468 - 0 .5927 - 4 . 162 0 .0002 Independent 0 . 1118 0 . 2713 1 . 594 0 . 1207 variables Coefficient Std . Error Ipartial t P pr7 0 .1782 25 pr10 0 . 2466 0 .08267 0 .4664 2. 983 0. 0054 ( Constant ) 9 .0610 pr11 0 .3134 0 .1393 0 . 3695 2 .249 0 .0315 pr4 12 .3884 3 . 2678 0 . 5756 3 . 791 0 . 0007 pr15 - 0 . 1211 0 .08413 - 0 .2465 - 1 .439 0 .1598 pr5 - 18 . 7518 6 . 3679 - 0 .4798 - 2 . 945 0 . 0063 pro 6 . 3645 3 . 1861 0 . 3478 1 . 998 0 . 0552 F - ratio 4 . 6613 pr12 14 . 3572 4 . 6488 0 .4975 3 . 088 0 .0044 Significance P = 0 .002 pr13 4 . 1192 2 . 3607 0 .3082 1 .745 0 .0916 30 level pr14 - 9 .8338 4 . 4575 - 0 . 3791 - 2 . 206 0 . 0354 prl - 4 . 5430 3 .0234 - 0 . 2688 - 1 . 503 0 . 1438 prl1 22 . 4197 4 . 2262 0 . 7018 5 . 305 < 0 . 0001 pr15 5 . 8245 3 .0380 0 .3354 1. 917 0 . 0651 F - ratio 6 . 4729 TABLE 39 Significance P < 0 .001 35 level Multiple regression Dependent Y avg _ PDT' PDT This relationship was also significantly affected by plasma avg PDT /PDT Sample size 39 homocysteine levels ( Table 37 ). Coefficient of 0 .5013 40 determination R ? TABLE 37 R2- adjusted 0 .4078 Multiple regression Regression Equation Dependent Y avg _ PDT Independent avg PDT 45 variables Coefficient Std . Error ( partial t P Sample size 39 Coefficient of 0 .6897 ( Constant ) 1 . 5047 determination R2 pr5 - 0 .5644 0 . 1456 - 0 . 5653 - 3 . 877 0 .0005 pr7 0 .2353 0 . 1084 0 . 3583 2 . 171 0. 0375 Regression Equation pr3 - 0 . 1677 0 . 09174 - 0 . 3074 - 1 . 827 0 . 0770 50 pr10 0 .2618 0 .08455 0 .4801 3 .096 0 . 0041 Independent pr11 0 .2222 0 . 1458 0 .2602 1 .525 0 . 1372 variables Coefficient Std . Error ( partial t P LHcy - 0 .02361 0 .01068 - 0 . 3638 - 2 . 209 0. 0344 F - ratio 5 . 3610 ( Constant ) 4 . 1698 Significance P = 0 .001 pr4 10 . 4761 3 . 4882 0 . 4936 3 . 003 0 . 0056 level pr5 - 18 .3444 6 . 2689 - 0 . 4839 - 2 .926 0 .0067 55 - pro 7 . 1093 3 . 1775 0 .3894 2 . 237 0 .0334 pr12 12 .5726 4 . 7438 0 .4478 2 . 650 0 .0131 pr13 4 . 3352 2 . 3266 0 . 3321 1 . 863 0 .0729 The baseline length of the G1 time in the lymphocyte pr14 - 9 .7313 4 . 3841 - 0 .3868 - 2 .220 0 .0347 cultures was significantly associated with a combination of pr1 - 6 . 0997 3 . 1717 - 0 .3416 - 1 . 923 0 .0647 SNPs on the rapamycin - sensitive genes (pr4 , pr5 , pr6 , pr12 , prl1 22 .9197 4 . 1712 0 .7203 5 . 495 < 0 .0001 pr15 5 .8184 2 . 9876 0 3454 1 947 00616 60 pr14 , prl , pr10 , prll and pr15 ) ( Table 40 ) . LHcy 0 .4774 0 . 3386 0 . 2574 1 .410 0 . 1697 F -ratio 6 . 2226 TABLE 40 Significance P < 0 . 001 level Multiple Regression 65 Dependent Y avg_ TG1 The change induced in cell proliferation (PDT ) by avg TG1 rapamycin was also significantly associated with a combi US 9, 944 ,986 B2 101 102 TABLE 40 - continued TABLE 42 42 . 1 Logistic regression Sample size 39 Coefficient of 0 .6827 Dependent Y DG _ AD determination R ? Select LHcy > 0 Sample size 39 Cases with Y = Control 20 (51 . 28 % ) Regression Equation Cases with Y = AD 19 48 .72 % )
Independent 10 42. 2 OVERALL MODEL FIT variables Coefficient Std . Error (partial t P Null model - 2 Log Likelihood 54 . 040 Significance level P = 0 .0162 ( Constant ) 7 .2421 15 42 . 3 COEFFICIENTS AND STANDARD ERRORS pr4 9 .7224 2 . 0230 0 .6658 4 .806 <0 .0001 pr5 - 8 .9847 4 . 3131 - 0 .3608 - 2 .083 0 .0462 Variable Coefficient Std . Error P pr6 3 . 8481 2 . 1043 0 . 3215 1. 829 0 .0778 prl = 1 - 1 . 35043 0 .90923 0 . 1375 pr12 10 . 2202 3 . 0217 0 .5319 3. 382 0 .0021 pr10 = 1 1 .69245 0 . 97968 0 . 0841 pr14 - 8 . 8697 3 . 0202 0 .0064 20 LHcy 0 . 35371 0 . 14382 0 . 0139 - 0. 4788 - 2. 937 Constant - 3 .9978 pri - 4 .4125 1 . 9865 - 0 .3813 - 2 . 221 0 .0343 pr10 - 2 .0526 1 .4835 - 0 . 2488 - 1. 384 0 .1770 42 . 4 ODDS RATIOS AND 95 % CONFIDENCE INTERVALS pr11 12. 5716 2 . 7938 0 .6412 4 .500 0 .0001 Variable Odds ratio 95 % CI pr15 6 .6133 1. 9953 0. 5242 3. 314 0. 0025 25 F - ratio 6 . 9327 prl = 1 0 . 2591 0 . 0436 to 1 . 5398 Significance P < 0 .001 pr10 = 1 5 . 4328 0 .7963 to 37. 0631 LHcy 1 . 4243 level 1 .0745 to 1 .8881 30 42. 5 CLASSIFICATION TABLE (CUT -OFF VALUE P = The ' efficacy of Rapamycin ' in the lymphocyte cultures Predicted group was also calculated from the combined effect on cell pro liferation time and relative lengthening of the G1 time. This Actual group 0 1 Percent correct estimate was based on idealised cell culture models . The 35 yY =. Control 1515 5 75 .00 % resulting value was significantly associated with a combi Y = AD 7 12 63 . 16 % nation of pr4, pr12 , pr10 and pr15 ( Table 41) . Percent of cases correctly classified 69 . 23 % TABLE 41 42. 6 ROC CURVE ANALYSIS 40 Multiple regression Area under the ROC curve (AUC ) 0 . 774 Standard Error 0 .0769 Dependent Y efficiency _of _ Rapa_ from _ ideal _model 95 % Confidence interval 0 .612 to 0 .892 efficiency of Rapa from ideal model Sample size 39 Coefficient of 0 . 3972 45 In summary the data presented indicate that the rapamycin determination R ? response in peripheral lymphocytes from individual patients Regression Equation is the result of the combination of SNPs on the rapamycin sensitive genes . The data also show that this genetic asso Independent ciation is strongly dependent on an environmental risk factor variables Coefficient Std . Error partial t P 50 that has an influence on cell proliferation and cell cycle ( Constant) 0 .6211 characteristics, and is itself an independent risk factor for pr4 - 0 . 5339 0 . 2317 - 0 . 3676 - 2 . 305 0 . 0274 pr12 - 0 .4911 0 . 1739 - 0 .4359 - 2 . 824 0 . 0079 AD . pr10 0 . 4397 0 . 1636 0 .4186 2 . 687 0 .0111 The present invention is not to be limited in scope by the pr15 - 0 . 4562 0 . 1653 - 0 .4279 - 2 .761 0 .0092 55 specific embodiments described herein . Indeed , various F - ratio 5 .6012 modifications of the invention in addition to those described Significance P = 0 .001 herein will become apparent to those skilled in the art from level the foregoing description and accompanying figures . Such modifications are intended to fall within the scope of the 4 . 3 the Association of the Variants on Rapamycin - sensitive 60 appended claims. Moreover , all aspects and embodiments of Genes with AD . the invention described herein are considered to be broadly Logistic regression indicates that a combination of prl. applicable and combinable with any and all other consistent pr10 and plasma homocysteine levels are significantly asso embodiments , including those taken from other aspects of ciated with the diagnosis of AD . The prediction from the 65 the invention ( including in isolation ) as appropriate . model would allow the correct classification of 77 .4 % of the Various publications are cited herein , the disclosures of patients ( AUC ) ( Table 42) . which are incorporated by reference in their entireties . US 9 ,944 ,986 B2 103 104
SEQUENCE LISTING
< 160 > NUMBER OF SEQ ID NOS : 16 < 210 > SEQ ID NO 1 < 211 > LENGTH : 18 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223???? > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 1 tcagctgtgg ggtcctgt 18
< 210 > SEQ ID NO 2 < 211 > LENGTH : 19 < 212 > TYPE : DNA < 2 13 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 2 gaaggggaca ggcagtgag 19
< 210 > SEQ ID NO 3 < 211 > LENGTH : 18 < 212 > TYPE : DNA ? 2 13 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 3 gatggcgact gtcgaacc 18
< 210 > SEQ ID NO 4 < 211 > LENGTH : 20 ?< 212 > TYPE : DNA ?< 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 4 tgggttagca acctcctgat 20
< 210 > SEQ ID NO 5 < 211 > LENGTH : 20 < 212 > TYPE : DNA ?< 213 > ORGANISM : Artificial Sequence ?< 220 > FEATURE : ?< 223NNPP > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 5 gtgtagcgca cactttctgg 20
< 210 > SEQ ID NO 6 < 211 > LENGTH : 20 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223NNA > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 6 tgggttagca acctcctgat 20
< 210 > SEQ ID NO 7 < 211 > LENGTH : 18 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence US 9, 944 ,986 B2 105105 106 - continued < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 7 ccgtgacctc aagccttc 18
< 210 > SEQ ID NO 8 < 211 > LENGTH : 18 < 212 > TYPE : DNA ?< ?213 > ORGANISM : Artificial Sequence ? ?< 220 > FEATURE : ? ?< 223 > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 8 gccaggccaa agtcacag 18
< 210 > SEQ ID NO 9 < 211 > LENGTH : 20 < 212 > TYPE : DNA ? 13 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 9 gcgaaggaca gtggagaagt 20
< 210 > SEO ID NO 10 < 211 > LENGTH : 19 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 10 gagagggcgg atggagata 19
< 210 > SEQ ID NO 11 < 211 > LENGTH : 19 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 11 ttgtgccgcg taagacagt 19
< 210 > SEO ID NO 12 < 211 > LENGTH : 20 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220NMOM > FEATURE : < 223 > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 12 cagcgtcagt gtcaggaagt 20
< 210 > SEO ID NO 13 < 211 > LENGTH : 21 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence ? 20 > FEATURE : < 223 > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 13 tgcccaagat ctgatacaag g 21 US 9, 944 ,986 B2 107 108 - continued
< 210 > SEQ ID NO 14 ?.< 211 > LENGTH : 20 ?< 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 14 ctccaacaca ccaccataca 20
< 210 > SEQ ID NO 15 < 211 > LENGTH : 19 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 15 ctcctggttc tgcccaagt 19
< 210 > SEQ ID NO 16 < 211 > LENGTH : 20 V< 212 > TYPE : DNA V< 213 > ORGANISM : Artificial Sequence V< 220 > FEATURE : V< 223 > OTHER INFORMATION : Synthetic primer < 400 > SEQUENCE : 16 caggttctct aggggcttcc 20
The invention claimed is : rs 1148613 , an A for rs1341665 , a G for rs290258 and 1 . A method for detection of a combination of human a T for rs2036108 . single nucleotide polymorphisms (SNPs ) in a human sub - > 2 . The method of claim 1 , further comprising detection of ject, comprising one or more SNP alleles selected from rs798893 (C ) , a ) obtaining a nucleic acid sample from said human rs1532278 ( C ) , rs1801274 ( G ) , rs811925 ( G ) , rs883524 ( C ) , subject; b ) genotyping the sample for a combination of SNPSs in rs 1065457 ( G ) , rs295 ( C ) , rs365836 ( G ) , rs1359059 ( A ) , and rapamycin - sensitive gene( s) FAM5C , CRP , SYK , and 4010 rs569214 ( T ). ADRAIA , said combination of SNP alleles being 3 . The method of claim 2 , wherein rs798893 ( C ) is rs725106 ( A ), rs 1148613 (C ), rs 1341665 ( A ), rs290258 detected . ( G ) and rs2036108 ( T ) ; and 4 . The method of claim 2 , where all SNP alleles are c ) detecting in said nucleic acid sample from said human detected . subject the presence of an A for rs725106 , a C for * * * * *